Materials and methods for altering an immune response to exogenous and endogenous immunogens, including syngeneic and non-syngeneic cells, tissues or organs

ABSTRACT

Disclosed herein are materials and methods for modulating an immunologically adverse response to an exogenous or endogenous immunogen, including a cell, tissue, or organ associated immunogen. An implantable material comprising cells, such as but not limited to endothelial cells, anchored or embedded in a biocompatible matrix can modulate an adverse immune or inflammatory reaction to exogenous or endogenous immunogens, including response to non-syngeneic or syngeneic cells, tissues or organs, exogenous immunogens or stimuli, as well as ameliorate an autoimmune condition. The implantable material can be provided prior to, coincident with, or subsequent to occurrence of the immune response or inflammatory reaction. The implantable material can induce immunological acceptance in a transplant patient, reduce graft rejection and reduce donor antigen immunogenicity.

FIELD OF THE INVENTION

This invention is directed to materials and methods for modulating animmunologically adverse response to an exogenous or endogenousimmunogen, including a cell-, tissue-, or organ-associated immunogen.For example, the present invention can modulate an adverse immuneresponse or inflammatory reaction to exogenous or endogenous immunogens,including non-syngeneic or syngeneic cells, tissues or organs, as wellas ameliorate an autoimmune condition.

BACKGROUND OF THE INVENTION

Research on xenotransplantation has been intensified over the past yearsto alleviate organ shortage. However, host immune responses present aformidable barrier to transplantation across species. Whereas naturalantibodies cause immediate rejection of such discordant transplants,endothelial cell (EC) injury and activation of graft vessel lining ECplay a pivotal role in initiating chronic graft rejection. Disruption ofthe integrity of the endothelial layer is of undoubted importance innumerous conditions, including syngeneic and non-syngeneic tissuetransplants as well as infectious, neoplastic, inflammatory andcardiovascular diseases.

Heretofore immunomodulation and transplant acceptance have requiredreliance on systemically-administered immunosuppressive agents. Whilesuch agents permit some degree of transplant acceptance, success islimited and perhaps of more significance, a patient's immune system isthoroughly compromised as a result of such agents. Thus a need stillremains for therapeutic materials and treatment paradigms which canachieve immunomodulation absent the toxicity and adverse affects on apatient's immune system.

Similarly, exogenous immunogens or stimuli have posed a clinicalchallenge. These, too, can result in adverse immunological events orinflammatory reactions which necessitate treatment. Heretofore, clinicalmanagement of such adverse events has relied almost exclusively ontreatments with pharmaceutical agents which suppress the immune systemnon-specifically.

Autoimmune diseases and other similar diseases are yet another clinicalmanifestation of heightened inflammatory reactions or adverse immuneresponses. Successful management of such diseases has eluded cliniciansto date.

An object of the present invention is to provide a tissue engineeringsolution for achieving immunomodulation without reliance on chemicals orpharmaceuticals which compromise a patient's immune system. This tissueengineering solution can be employed to alter, in a clinically practicalmanner, an immune response to exogenous and endogenous immunogens,including non-syngeneic as well as syngeneic cell-, tissue- ororgan-associated immunogens. Another object of the present invention isto facilitate a patient's acceptance of non-syngeneic as well assyngeneic cells, tissues or organs. Another object of the presentinvention is to employ this tissue engineering solution to modulate aninflammatory reaction such as that associated with injury and variousdiseases. Another object is to utilize the materials and methods of thepresent invention to manage autoimmunity and related diseases.

SUMMARY OF THE INVENTION

The present invention exploits the discovery that cells anchored toand/or embedded within a biocompatible matrix, preferably one having athree-dimensional configuration, can modulate an immunologically adverseresponse or inflammatory reaction to any exogenous or endogenousimmunogen. Immunogen includes any syngeneic or non-syngeneic immunogen,including a cell-, tissue-, or organ-associated immunogen, as well asinjury, disease and environmental stimuli.

In one aspect, the present invention is a method of reducing an immuneresponse or an inflammatory reaction. According to this method, arecipient is provided an implantable material comprising a biocompatiblematrix and anchored and/or embedded endothelial cells, endothelial-likecells, or analogs thereof. The implantable material is provided to therecipient in an amount sufficient to reduce the immune response orinflammatory reaction in the recipient.

According to the invention, the providing step can occur prior to,coincident with, or subsequent to administration to the recipient of oneor more doses of a cell, tissue or organ from a syngeneic ornon-syngeneic donor. According to another embodiment, the providing stepis prior to, coincident with, or subsequent to occurrence of an immuneresponse or inflammatory reaction. According to another embodiment, themethod reduces an immune response or an inflammatory response bymodulating immunological memory.

In a related aspect, the present invention is a method of inducingimmunological acceptance in a patient. According to this method, thepatient is provided an implantable material comprising a biocompatiblematrix and anchored and/or embedded endothelial cells, endothelial-likecells, or analogs thereof, prior to, coincident with, or subsequent totransplantation of autograft, xenograft or allograft cells, tissue ororgan in an amount effective to induce acceptance in the patient.

Additionally, the present invention is directed to a method of reducingdonor antigen immunogenicity. According to this method, an implantablematerial comprising a biocompatible matrix and anchored and/or embeddedendothelial cells, endothelial-like cells, or analogs thereof arepresented prior to, coincident with, or subsequent to introduction ofthe donor antigen to a recipient in an amount effective to reduce donorantigen immunogenicity. According to another embodiment, the donor andrecipient are the same. According to a further embodiment, the recipienthas an autoimmune disease. According to yet another embodiment, thedonor antigen comprises a non-endothelial cell antigen.

According to various other embodiments, the providing step occurs priorto, coincident with, or subsequent to administration to the recipient ofan immunosuppressive agent. The immunosuppressive agent can reside inthe implantable material.

Moreover, the present invention is also directed to a method oftransplanting to a recipient a syngeneic or non-syngeneic cell, tissueor organ transplant. According to this method, a recipient is providedan implantable material comprising a biocompatible matrix and anchoredand/or embedded endothelial cells, endothelial-like cells, or analogsthereof, prior to, coincident with, or subsequent to transplantationsuch that the transplanted syngeneic or non-syngeneic cell, tissue ororgan is not rejected by the recipient. According to one embodiment ofthe method, the transplanted cell, tissue or organ comprisesnon-endothelial cells.

In another aspect, the present invention is an implantable materialcomprising a biocompatible matrix and cells anchored thereto and/orembedded therein. According to one currently preferred embodiment, thecells are endothelial cells, endothelial-like cells and/or analogs ofeither. In certain other embodiments, endothelial-like cells or analogsof the implantable material are non-endothelial cells. According toanother embodiment, the cells of the implantable material are autogenic,allogenic, xenogenic or genetically-modified variants of any one of theforegoing cell types. According to a further preferred embodiment, thecells of the implantable material are vascular endothelial cells.According to one embodiment, the implantable material is a solid ornon-solid. According to yet another, the implantable material isprovided to the recipient by implantation, injection or infusion.

The present invention is also directed to an implantable material forreducing an immune response to a syngeneic or non-syngeneic cell, tissueor organ. According to this aspect of the invention, the implantablematerial comprises a biocompatible matrix and, anchored thereto and/orembedded therein, endothelial cells, endothelial-like cells, or analogsthereof. According to this aspect of the invention, an effective amountof the implantable material reduces the recipient's immune response tothe syngeneic or non-syngeneic cell, tissue or organ. According to oneembodiment of this aspect of the present invention, the cell, tissue ororgan is that of the recipient suffering from an autoimmune disease.

The invention is also directed to a variation of the above-describedimplantable material which is useful for reducing an immune response toa non-syngeneic cell, tissue or organ, wherein said implantable materialcomprises cells, tissue, or organ or a segment thereof anchored toand/or embedded within the biocompatible matrix. An effective amount ofthis implantable material reduces the recipient's immune response to anon-syngeneic cell, tissue or organ. The non-syngeneic cell, tissue ororgan is that of the recipient suffering from an autoimmune disease.

In a further aspect, the present invention is a cell suitable for usewith the implantable material of any one of inventions described herein.According to one embodiment, the endothelial-like cell or its analog isa non-endothelial cell. According to another embodiment, the analog isnon-natural. According to a further embodiment, the cell, when anchoredto and/or embedded within a biocompatible matrix, reduces a recipient'shumoral or cellular immune response to a syngeneic or non-syngeneicdonor cell, tissue or organ.

According to another embodiment, the cell, when anchored to and/orembedded within a biocompatible matrix, exhibits diminishedimmunogenicity. According to one embodiment, the cell exhibitsdiminished immunogenicity by exhibiting reduced expression of MHC orreduced capacity to bind, activate or promote maturation of innateimmune cells, when anchored to and/or embedded within a biocompatiblematrix, wherein said innate immune cells are selected from the groupconsisting of NK cells, dendritic, cells, monocytes, and macrophages.

According to another embodiment, the cell, when anchored to and/orembedded within a biocompatible matrix, exhibits reduced expression ofcostimulatory molecules or adhesion molecules. According to a furtherembodiment, the cell, when anchored to and/or embedded within abiocompatible matrix and co-cultured with a dendritic cell, inhibitsexpression by said dendritic cell of HLA-DR, IL12, Toll-like receptor orCD83; promotes uptake of dextran by said dendritic cell; or blocksdendritic cell-induced lymphocyte proliferation; or when co-culturedwith adaptive immune cells inhibits proliferation, activation ordifferentiation of said cells, wherein adaptive immune cells areselected from the group consisting of B-lymphocytes and T-lymphocytes.

In another aspect, the present invention is a cell bank comprising anyone of the cells described herein. In a further aspect, the presentinvention is a bank comprising any one of the implantable materialsdescribed herein.

In a further aspect, the present invention is a method of reducing animmune response or an inflammatory reaction resulting from exposure toan exogenous immunogen. According to this method, a recipient isprovided with an implantable material comprising a biocompatible matrixand anchored or embedded endothelial cells, endothelial-like cells, oranalogs thereof. The implantable material is provided to the recipientin an amount sufficient to reduce the immune response or inflammatoryreaction in the recipient resulting from exposure to the exogenousimmunogen.

According to one embodiment of this method, the providing step is priorto, coincident with, or subsequent to occurrence of the immune responseor inflammatory reaction. According to another embodiment, the exogenousimmunogen is naturally occurring. According to a further embodiment, theexogenous immunogen is selected from the group consisting ofpharmaceutical agents, toxins, surgical implants, infectious agents andchemicals. According to another embodiment, the exogenous immunogen isan exogenous stimulus selected from the group consisting ofenvironmental stress, injury and exposure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 1C graphically depict levels of circulatingPAE-specific antibodies according to an illustrative embodiment of theinvention.

FIG. 2 graphically depicts lytic activity of splenocytes according to anillustrative embodiment of the invention.

FIG. 3A graphically depicts the frequencies of cytokine-producing cellsaccording to an illustrative embodiment of the invention.

FIG. 3B depicts representative ELISPOT wells according to anillustrative embodiment of the invention.

FIG. 3C graphically depicts the frequencies of T cells according to anillustrative embodiment of the invention.

FIGS. 4A and 4B graphically plot levels of effector cells according toan illustrative embodiment of the invention.

FIGS. 5A, 5B and 5C graphically depict antibody levels according to anillustrative embodiment of the invention.

FIG. 6 graphically depicts levels of splenocytes according to anillustrative embodiment of the invention.

FIGS. 7A and 7B graphically depict antibody levels according to anillustrative embodiment of the invention.

FIGS. 8A and 8B graphically depict the frequency of cytokine-producingcells according to an illustrative embodiment of the invention.

FIGS. 9A and 9B graphically depict effector cell levels according to anillustrative embodiment of the invention.

FIGS. 10A and 10B depict correlations between the frequency ofTh2-cytokine producing splenocytes and the extent of T celldifferentiation into effector cells according to an illustrativeembodiment of the invention.

FIG. 11 graphically depicts the degree of damage to endothelial cellsaccording to an illustrative embodiment of the invention.

Figures which refer to “embedded” or “matrix-embedded” PAE, HAE or ECmean matrix-anchored and/or matrix-embedded PAE, HAE, EC.

DETAILED DESCRIPTION OF THE INVENTION

Tissue engineering is a promising approach to exploit endothelial cells,endothelial-like cells, or analogs of either as a cellular therapy fordiseases accompanied by or typified by adverse immunological components.For example, certain diseases such as but not limited to vasculardiseases provoke adverse immunological responses and/or inflammatoryreactions. The present invention is based on the discovery that cellssuch as endothelial cells which are anchored to or embedded inthree-dimensional matrices, secrete essential regulatory factors whichcan ameliorate or otherwise modulate an adverse immunological response.

The implantable material of the present invention was developed on theprincipals of tissue engineering and represents a novel approach toaddressing the herein-described clinical needs. The implantable materialof the present invention is unique in that the viable cells anchored toand/or embedded within the biocompatible matrix are able to supply tothe site of administration multiple cell-based products in physiologicalproportions under physiological feed-back control. As describedelsewhere herein, the cells suitable for use with the implantablematerial are endothelial, endothelial-like cells, or analogs of each ofthe foregoing. Local delivery of multiple compounds by these cells andphysiologically-dynamic dosing provide more effective regulation of theprocesses responsible for modulating an immune response. The implantablematerial of the present invention can provide an environment whichmimics supportive physiology and is conducive to modulation of an immuneresponse.

This is an unexpected discovery since endothelial cells can play apivotal role in initiation of adverse allo- and xeno-immune responses.Moreover, endothelial cells can activate T-cells throughantigen-mediated processes and T-cell activation can modify crucialendothelial cell function, including antigen presentation via activationby cytokines, thereby contributing to an adverse immune response. And,endothelial cells constitutively express class I MajorHistocompatibility Complex (MHC) molecules, and IFN-γ can induceendothelial cells to express class II MHC molecules which allows them toprovide antigen-dependent signals to CD8⁺ and CD4⁺ T-cells through thedirect pathway. Endothelial cells also can primarily providecostimulation to T-cells. In addition, the capacity to capture T-cellsvia endothelial expression of adhesion molecules allows formation ofcontact regions which furthers the adverse immune response in the formof inflammation. Furthermore, autoimmunity can exacerbate vasculardisease, in particular in the form of anti-endothelial cell antibodies.The heightened morbidity of cardiovascular diseases in concert withdiabetes mellitus, hypertension and other disease states reflects theincreased presence and potency of these antibodies.

In contrast, as disclosed herein, matrix-anchored and/or -embeddedendothelial cells, when implanted in a host, act as powerful regulatorsof the immune system as indicated by significant reduction in theexpected systemic immune response and/or local inflammation. Asexemplified herein, the ability of such cells to ameliorate or modulateimmune responsiveness has been demonstrated by comparing the immuneresponse against free versus matrix-anchored and/or -embeddedendothelial cells in naïve mice as well as mice with heightenedendothelial cell immune reactivity. Matrix-associated endothelial cellsas described herein provide immune protection at multiple levels; humanand porcine endothelial cells demonstrate a marked reduction inelaborated MHC class molecules; costimulatory molecules; and adhesionmolecules when matrix-anchored and/or -embedded as disclosed herein.

Matrix anchoring and/or embedding of endothelial cells can alsoinfluence formation of immunological memory as exemplified herein.Whereas reimplantation of free, saline-suspended endothelial cellpellets alone or as pellets situated adjacent to an empty matrix evokeda significant increased humoral and cellular xenoresponse, rechallengingmice with matrix-anchored and/or -embedded endothelial cells led to areduced lytic ability of splenocytes without enhancing the humoralimmune responses. Moreover, a modest shift in the Th1/Th2 balancetowards the former was obvious in mice receiving matrix-anchored and/or-embedded xenogeneic endothelial cells.

Thus, introduction of free endothelial cells adjacent to an empty matrixfailed to reduce the host immune response indicating the importance ofmatrix-anchoring and/or -embedding. Failure of anchored and/or embeddedendothelial cells to express MHC II, costimulatory, and adhesionmolecules upon stimulation could account for the attenuateddifferentiation of T-cells in effector cells in response to implantedmatrix-anchored and/or -embedded xenogeneic endothelial cells. Asexplained herein, activation of mice splenocytes is muted when exposedto matrix-anchored and/or -embedded xenogeneic endothelial cells in aMHC class II dependent manner.

Overall the isotropic nature of endothelial cells contributes to thisunique form of immunomodulation wherein cell anchoring and/or embeddingin a suitable matrix provides immunoprotection through isolation ormasking of critical antigens. It is well recognized that in vivoendothelial cell function is anchorage- and density-dependent. Previousstudies have shown that the endothelial basement membrane (EBM) controlsaspects of cell adhesion, spreading, migration, contractility,differentiation, proliferation, protein synthesis and secretion.Furthermore, EBM is altered in many in vivo disease states, fromdiabetes to glomerulopathy to atherosclerosis. Dysfunction ofendothelial cells correlates with changes in basement membranecomposition cumulating in the degree of attachment of endothelial cells,and the quality of basement membrane anchoring plays a role forendothelial cells immunobiology.

The present invention is based on the unexpected discovery thatanchoring and/or embedding endothelial cells in a suitable biocompatiblematrix, such as but not limited to a 3-dimensional collagen-basedmatrix, can transform xenogeneic endothelial cells into animmunologically non-offending cell phenotype. Such a discovery can nowbe exploited by the skilled practitioner, following the guidanceprovided herein, as a tolerance-inducing approach to syngeneic ornon-syngeneic therapies such as but not limited to allotransplantationor xenotransplantation as exemplified herein. For example, in apreferred embodiment of the present invention, a clinician can diminishand/or delay rejection by implanting matrix-anchored and/or -embeddedendothelial cells prior to transplantation of an allo- or xenografttissue or organ. For purposes of the present invention, blood is a typeof tissue.

Pre-treatment acclimates the recipient's immune system and can result ina reduced, attenuated and/or delayed immune response to a graft. Thepresent invention does not require that the implantable materialcomprise anchored- and/or embedded cells which are the same as orsimilar to those ultimately transplanted in the recipient. All that isrequired is that the implantable material comprising anchored- and/orembedded cells has an immunomodulatory effect when provided to arecipient. In certain circumstances, a single administration prior to orcoincident with a transplant can be sufficient. In other circumstances,multiple or serial administrations are preferred. The skilled clinicianwill recognize such circumstances.

As is well recognized, even transplantation of allogeneic cells is oftenaccompanied by an immune response. A question of much interest iswhether this is a constitutive and immutable property of foreign cellsor one that can be regulated. The experiments set forth hereindemonstrate that the immunogenicity of cells that are normally anchoredto basement membranes can be markedly reduced if implanted in amatrix-anchored and/or -embedded state an effect not seen when thesesame cells were injected in a free state. Other experiments set forthherein investigate the influence of heightened anti-endothelial cellimmunity which is a common clinical feature in a variety of autoimmuneand endocrinological diseases.

Additionally, certain of the experiments summarized herein demonstratethat serial injections of free porcine aortic endothelial cells (PAE)induced circulating anti-PAE antibodies, elevating immunosensitivity.The response to subsequent PAE injections was even greater than thatobserved upon first exposure. In contrast, when PAE were implanted in amatrix-anchored and/or -embedded state, the immune response tosubsequent exposures was muted and dropped significantly over time.Also, as illustrated below, the initial response to endothelial cells isIgM-mediated, lower than the subsequent IgG response and muted whenpreceded by serial injections. The IgM response is more evident in naïvethan pre-sensitized animals and takes longer to abate after free PAEexposure to than after exposure to matrix-anchored and/or -embeddedendothelial cells.

Pre-sensitization of mice with suspensions of free PAE resembles theIgG₁-driven anti-endothelial immunity seen in diabetes mellitus,hypertension and autoimmune diseases. The cellular immune response tofree and matrix-anchored and/or -embedded cells followed the pattern ofhumoral immunity. Repeated exposure to antigens resulted in increasedformation of memory and subsequently in a more vigorous immune reactionby effector T cells. Hence, the induction of xenoreactive IL-4- andIL-10-producing splenocytes and effector T cells was elevated over timeand visible after implantation of free endothelial cells in naïve andpre-sensitized mice. In all mice, cytokine levels correlated linearlyand precisely with effector T cell induction further supporting thenotion of a Th2-driven cellular response in xenoreactivity andaccentuating the immunosilencing aspects of matrix-embedded endothelialcells to activate adaptive immune mechanisms. Damage to implantedendothelial cells correlated with the extent of the immune responseelicited. Implanted cells were most profoundly affected afterpre-sensitization and with free PAE. The decreased induction of humoraland cellular immune responses in naïve mice receiving matrix-embeddedendothelial cells resulted in a lesser degree of damage by host immunecells.

These experiments provide insights into the activation of and damage toendothelial cells, suggesting a pivotal role for cell-matrix contact.The honeycomb-like structure of a currently preferred matrix, Gelfoam,allows endothelial cells to associate with, or anchor to, or embedwithin its three-dimensional configuration and in certain embodiments,line the internal surfaces of this matrix in a fashion which simulatesthe appearance of confluent endothelium in quiescent vessels. Thus incertain embodiments, anchoring to and/or embedding endothelial cellswithin a matrix with the properties of Gelfoam resembles the physiologicthree-dimensional state of intact endothelium. The experiments set forthbelow demonstrate that matrix-anchoring and/or embedding not onlyprotects endothelial cells from host immune reactions but changes thehost's perception of endothelial cell immunogenicity.

Thus, during disease for example, phenotypic transformation ofendothelial cells dislocated from an intact, matrix-adherent endogenousstate to a free state is likely critical to initiation and perpetuationof vascular disease, for example. The teachings herein indicate thatendothelial cell detachment precedes expression of adhesion,costimulatory and MHC molecules which is then followed by attraction ofimmune cells, perpetuating endothelial activation and cell damage. Inthis regard, the immunobiological and immunoreactive qualities ofendothelial cells correlate with morphology and function. Endothelialcells from different vascular beds and divergent basement membraneconnectivity demonstrate marked differences in constitutive andinducible expression of adhesion, costimulatory and MHC-molecules.Further, there is growing appreciation that deposition of transitionalextracellular matrix proteins such as fibronectin and fibrinogen intothe subendothelial matrix as well as detachment of endothelial cellsfrom the basement membrane affects intra-endothelial cell signaling.

As contemplated by the present invention, manipulation of cellphenotype, immunogenicity, and function can be used to tailor theproperties of tissue engineered constructs developed in vitro forregenerative purposes; in particular, such a use of the presentinvention is clinically beneficial since current cell-based therapiesare limited by profound host immune reactions. For example, the presentinvention is particularly useful for treatment of atheroscleroticdisease since the presence of activated immune cells and inflammationare key pathophysiologic components. Similarly, heightenedanti-endothelial immunity has been identified as a pivotal rate-limitingeffect for endothelial cell-based therapies, such as but not limitedtherapies involving seeding of the interior of a vascular structure withcells or tissue. In contrast, the present invention can be exploited tomanage endothelial cell phenotypic shifts which occur in vascularpathology, e.g., via dearrangement of cell-matrix contact, andappropriately targeted therapeutic options can then be implemented inthe clinic using the materials and methods of the present invention.

Taken together, the teachings presented herein also demonstrate thatfeatures of a matrix such as but not limited to biocompatibility,porosity, three-dimensionality, can support the growth of a populationof endothelial cells and can modulate the immunogenicity of such cells.Endothelial cells anchored to and/or embedded within a three-dimensionalmatrix elicited far less activation of host immune mechanisms and weresubject to far lower attack and damage from host immune cells. Findingsin naive mice were amplified in hosts with heightened anti-endothelialimmunity. In vivo studies presented herein show a marked decrease in theTh2-driven immune response in animals implanted with a matrix such asGelfoam comprising anchored and/or embedded endothelial cells versusanimals injected with free endothelial cells. In order for endothelialcells to activate naïve host T-cells, two signals are required: 1)antigen-presentation in the context of MHC molecules expressed on thedonor endothelial cells and 2) a second signal provided by acostimulatory molecule also expressed on the donor endothelial cellsurface. Therefore, while not wishing to be bound by theory, onepossible explanation for the observed results is that the interactionbetween a biocompatible matrix and embedded endothelial cells results ina decrease in surface expression of crucial costimulatory, MHC and/oradhesion molecules on the donor endothelial cells. Indeed, in vitroanalysis of critical adhesion, MHC-II and co-stimulatory moleculeexpression on both PAE and HAE (human aortic endothelial cells) show amatrix-anchored and/or embedded dependent profile. The expressionprofiles of adhesion (E-selectin, P-selectin, ICAM-1, VCAM-1, and CD58),costimulatory (CD40, CD80, CD86) and MHC-II molecules were all reducedin endothelial cells anchored to and/or embedded with a Gelfoam matrixas compared to the same endothelial cells grown on standard tissueculture plates. P-selectin, E-selectin and VCAM-1 are closely associatedwith T-cell recruitment at sites of immune inflammation. Because antigenpresentation to CD4+ T-cells via MHC class II molecules is essential forhost immune recognition in the setting of non-vascularized xenogeneicimplants, the observed reduced MHC-II expression on matrix-anchoredand/or -embedded endothelial cells translated into a reducedproliferative response of host splenocytes. Furthermore, repeated invitro exposure of the same splenocytes to endothelial cells grown ontissue culture plates elicited a more vigorous secondary response,whereas there was no increased secondary response and therefore nomemory of prior exposure to matrix-anchored and/or -embedded endothelialcells. These in vitro findings correlate with the significantly mutedimmune reaction observed in rats and mice after implantation andre-challenge with matrix-anchored and or embedded endothelial cells asexemplified herein.

Similarly, a mechanism by which culturing endothelial cells in abiocompatible matrix such as but not limited to Gelfoam affectsexpression of MHC class II molecules, and subsequent endothelialimmunogenicity in vitro, was further elucidated by investigatingintracellular signaling pathways. Endothelial expression of MHC class IImolecules is induced by proinflammatory cytokines (e.g. interferon(IFN)-γ) that are secreted by activated immune cells (e.g. T-cells).Binding of proinflammatory cytokines to their receptors on endothelialcells initiates an intracellular signaling cascade resulting inphosphorylation of Janus protein tyrosine kinase (e.g. JAK-1 and 2) andsignal transducer and activators of transcription (e.g. STAT-1).Activation of JAK and STAT are usually tightly regulated within a targetcell. As set forth below, detailed in vitro analyses demonstrateddifferences in IFN-γ induced intracellular signaling pathways betweenendothelial cells grown to confluence on tissue culture plates ascompared to those anchored to and/or embedded or within Gelfoammatrices. Gelfoam-embedded HAE exhibited lower rates ofSTAT-phosphorylation and activation of the crucial interferon-regulatoryfactor-1 (IRF-1) with no change in surface IFN-γ receptor expression.Lower rates of JAK activation were also seen upon stimulation of HAE inGelfoam with IFN-γ.

Upon further investigation, it was observed that non-IFN-γ stimulatedHAE grown on a Gelfoam matrix expressed significantly higher levels ofthe counteracting inhibitory molecule, Suppressor of Cytokine Signaling(SOCS)-1 and 3, than HAE grown on tissue culture plates. One explanationtherefore for the muted IFN-γ induced intracellular signaling inGelfoam-embedded HAE is that the increased levels of SOCS-1 and 3resulted in an increase in the threshold for cytokine-induced activationof endothelial cells.

In a currently preferred embodiment, the implantable material of thepresent invention comprising anchored and/or embedded endothelial cellsis implanted at any non-luminal site. Thus, immediate exposure of thedonor cells to the host circulation is not required. Recent evidence hasdemonstrated the importance of the soluble endothelial factor CX₃CL1(fractalkine) for attraction of immune cells (i.e., natural killercells) and surface expressed forms of fractalkine for adherence of thoseimmune cells. Given that only modest cellular infiltration in and aroundimplantation sites of xeno- and allogeneic matrix-anchored and/orembedded endothelial cells was observed, release of soluble and surfaceexpression of fractalkine on HAE was quantified. As illustrated inexperiments set forth below, matrix-anchored and/or embedded endothelialcells showed reduced secretion and down-regulation of fractalkinesurface expression upon cytokine stimulation as compared to HAE grown ontissue culture plates. This resulted in significantly less adherence ofhuman natural killer cells to matrix-anchored and/or embedded HAE invitro.

Taken together, the changes in intracellular signaling, increased levelsof SOCS-1 and 3 (resulting in attenuated expression of NMC-II moleculesand subsequent T-cell activation) as well as reduced secretion andsurface expression of fractalkine in matrix-anchored and/or embeddedendothelial cells as compared to cells grown on tissue culture platesindicated an altered endothelial cell immunogenicity attributable tomatrix-embedding.

Cell Source. As described herein, the implantable material of thepresent invention comprises cells which can be syngeneic, allogeneic,xenogeneic or autologous. In certain embodiments, a source of livingcells can be derived from a suitable donor. In certain otherembodiments, a source of cells can be derived from a cadaver or from acell bank.

In one currently preferred embodiment, cells are endothelial cells. In aparticularly preferred embodiment, such endothelial cells are obtainedfrom vascular tissue, preferably but not limited to arterial tissue. Asexemplified below, one type of vascular endothelial cell suitable foruse is an aortic endothelial cell. Another type of vascular endothelialcell suitable for use is umbilical cord vein endothelial cells. And,another type of vascular endothelial cell suitable for use is coronaryartery endothelial cells. Yet other types of vascular endothelial cellssuitable for use with the present invention include pulmonary arteryendothelial cells and iliac artery endothelial cells.

In another currently preferred embodiment, suitable endothelial cellscan be obtained from non-vascular tissue. Non-vascular tissue can bederived from any tubular anatomical structure as described elsewhereherein or can be derived from any non-vascular tissue or organ.

In yet another embodiment, endothelial cells can be derived fromendothelial progenitor cells or stem cells; in still another embodiment,endothelial cells can be derived from progenitor cells or stem cellsgenerally. In a preferred embodiment, the cells can be progenitor cellsor stem cells. In other preferred embodiments, cells can benon-endothelial cells that are syngeneic, allogeneic, xenogeneic orautologous derived from vascular or non-vascular tissue or organ. Thepresent invention also contemplates any of the foregoing which aregenetically altered, modified or engineered.

In a further embodiment, two or more types of cells are co-cultured toprepare the present implantable material. For example, a first cell canbe introduced into the biocompatible matrix and cultured untilconfluent. The first cell type can include, for example, smooth musclecells, fibroblasts, stem cells, endothelial progenitor cells, acombination of smooth muscle cells and fibroblasts, any other desiredcell type or a combination of desired cell types suitable to create anenvironment conducive to endothelial cell growth. Once the first celltype has reached confluence, a second cell type is seeded on top of thefirst confluent cell type in, on or within the biocompatible matrix andcultured until both the first cell type and second cell type havereached confluence. The second cell type may include, for example,endothelial cells or any other desired cell type or combination of celltypes. It is contemplated that the first and second cell types can beintroduced step wise, or as a single mixture. It is also contemplatedthat cell density can be modified to alter the ratio of smooth musclecells to endothelial cells. Similarly, matrices can be seeded initiallywith a mixture of different cells suitable for the intended indicationor clinical regimen.

All that is required of the anchored and/or embedded cells of thepresent invention is that they exhibit one or more preferred phenotypesor functional properties. The present invention is based on thediscovery that a cell having a readily identifiable phenotype (describedelsewhere herein) when associated with a preferred matrix can reduce,ameliorate, and/or otherwise modulate an immune response or inflammatoryreaction via systemic and/or local effects.

For purposes of the present invention, one such preferred, readilyidentifiable phenotype typical of cells of the present invention is analtered immunogenic phenotype as measured by the in vitro assaysdescribed elsewhere herein. Another readily identifiable phenotypetypical of cells of the present invention is an ability to block orinterfere with dendritic cell maturation as measured by the in vitroassays described elsewhere herein. Each phenotype is referred to hereinas an immunomodulatory phenotype.

Evaluation of Immunomodulatory Functionality: For purposes of theinvention described herein, the implantable material can be tested forindicia of immunomodulatory functionality prior to implantation. Forexample, samples of the implantable material are evaluated to ascertaintheir ability to reduce expression of MHC class II molecules, to reduceexpression of co-stimulatory molecules, to inhibit the maturation ofco-cultured dendritic cells, and to reduce the proliferation of T cells.In certain preferred embodiments, the implantable material can be usedfor the purposes described herein when the material is able to reduceexpression of MHC class II molecules by at least about 25-80%,preferably 50-80%, most preferably at least about 80%; to reduceexpression of co-stimulatory molecules by at least about 25-80%,preferably 50-80%, most preferably at least about 80%; inhibitmaturation of co-cultured dendritic cells by at least about 25-95%,preferably 50-95%, most preferably at least about 95%; and/or reduceproliferation of lymphocytes by at least about 25-90%, preferably50-90%, most preferably at least about 90%.

Levels of expression of MHC class II molecules and co-stimulatorymolecules can be quantitated using routine flow cytometry analysis,described in detail below. Proliferation of lymphocytes can bequantitated by in-vitro coculturing ³[H]-thymidine-labeledCD3+-lymphocytes with the implantable composition viascintillation-counting as described below in detail. Inhibition ofdendritic cell maturation can be quantitated by either co-culturing theimplantable material with dendritic cells and evaluating surfaceexpression of various markers on the dendritic cells by flow cytometryand FACS analysis, or by measuring dendritic cell uptake ofFITC-conjugated dextran by flow cytometry. Each of these methods isdescribed in detail below.

In a typical operative embodiment of the present invention, cells neednot exhibit more than one of the foregoing phenotypes. In certainembodiments, cells can exhibit more than one of the foregoingphenotypes.

While the foregoing phenotypes each typify a functional endothelialcell, such as but not limited to a vascular endothelial cell, anon-endothelial cell exhibiting such a phenotype(s) is consideredendothelial-like for purposes of the present invention and thus suitablefor use with the present invention. Cells that are endothelial-like arealso referred to herein as functional analogs of endothelial cells; orfunctional mimics of endothelial cells. Thus, by way of example only,cells suitable for use with the materials and methods disclosed hereinalso include stem cells or progenitor cells that give rise toendothelial-like cells; cells that are non-endothelial cells in originyet perform functionally like an endothelial cell using the parametersset forth herein; cells of any origin which are engineered or otherwisemodified to have endothelial-like functionality using the parameters setforth herein.

Typically, cells of the present invention exhibit one or more of theaforementioned phenotypes when present in confluent, near-confluent orpost-confluent populations and associated with a preferred biocompatiblematrix such as those described elsewhere herein. As will be appreciatedby one of ordinary skill in the art, confluent, near-confluent orpost-confluent populations of cells are identifiable readily by avariety of techniques, the most common and widely-accepted of which isdirect microscopic examination. Others include evaluation of cell numberper surface area using standard cell counting techniques such as but notlimited to a hemocytometer or coulter counter.

Additionally, for purposes of the present invention, endothelial-likecells include but are not limited to cells which emulate or mimicfunctionally and phenotypically confluent, near-confluent orpost-confluent endothelial cells as measured by the parameters set forthherein.

Thus, using the detailed description and guidance set forth below, thepractitioner of ordinary skill in the art will appreciate how to make,use, test and identify operative embodiments of the implantable materialdisclosed herein. That is, the teachings provided herein disclose allthat is necessary to make and use the present invention's implantablematerials. And further, the teachings provided herein disclose all thatis necessary to identify, make and use operatively equivalentcell-containing compositions. At bottom, all that is required is thatequivalent cell-containing compositions are effective to modulate animmune response in accordance with the methods disclosed herein. As willbe appreciated by the skilled practitioner, equivalent embodiments ofthe present composition can be identified using only routineexperimentation together with the teachings provided herein.

In certain preferred embodiments, endothelial cells used in theimplantable material of the present invention are isolated from theaorta of human cadaver donors. Each lot of cells is derived from asingle or multiple donors, tested extensively for endothelial cellpurity, biological function, the presence of bacteria, fungi, knownhuman pathogens and other adventitious agents. The cells arecryopreserved and banked using well-known techniques for later expansionin culture for subsequent formulation in biocompatible implantablematerials. In other embodiments, living cells can be harvested from adonor or from the patient for whom the implantable material is intended.

Cell Preparation. As stated above, suitable cells can be obtained from avariety of tissue types and cell types. In certain preferredembodiments, human aortic endothelial cells used in the implantablematerial are isolated from the aorta of cadaver donors. In otherembodiments, porcine aortic endothelial cells (Cell Applications, SanDiego, Calif.) are isolated from normal porcine aorta by a similarprocedure used to isolate human aortic endothelial cells. Each lot ofcells is derived from a single or multiple donors, tested extensivelyfor endothelial cell viability, purity, biological function, thepresence of mycoplasma, bacteria, fungi, yeast, known human pathogensand other adventitious agents. The cells are further expanded,characterized and cryopreserved to form a working cell bank at the thirdto sixth passage using well-known techniques for later expansion inculture and for subsequent formulation as biocompatible implantablematerial.

The following is an exemplary protocol for preparing endothelial cellssuitable for use with the present invention. Human or porcine aorticendothelial cells are prepared in T-75 flasks pre-treated by theaddition of approximately 15 ml of endothelial cell growth media perflask. Human aortic endothelial cells are prepared in Endothelial GrowthMedia (EGM-2, Cambrex Biosciences, East Rutherford, N.J.). EGM-2consists of Endothelial Cell Basal Media (EBM-2, Cambrex Biosciences)supplemented with EGM-2 which contain 2% FBS. Porcine cells are preparedin EBM-2 supplemented with 5% FBS and 50 μg/ml gentamicin. The flasksare placed in an incubator maintained at approximately 37° C. and 5%CO₂/95% air, 90% humidity for a minimum of 30 minutes. One or two vialsof the cells are removed from the −160° C.-140° C. freezer and thawed atapproximately 37° C. Each vial of thawed cells is seeded into two T-75flasks at a density of approximately 3×10³ cells per cm³, preferably,but no less than 1.0×10³ and no more than 7.0×10³; and the flaskscontaining the cells are returned to the incubator. After about 8-24hours, the spent media is removed and replaced with fresh media. Themedia is changed every two to three days, thereafter, until the cellsreach approximately 85-100% confluence preferably, but no less than 60%and no more than 100%. When the implantable material is intended forclinical application, only antibiotic-free media is used in thepost-thaw culture of human aortic endothelial cells and manufacture ofthe implantable material of the present invention.

The endothelial cell growth media is then removed, and the monolayer ofcells is rinsed with 10 ml of HEPES buffered saline (HEPES). The HEPESis removed, and 2 ml of trypsin is added to detach the cells from thesurface of the T-75 flask. Once detachment has occurred, 3 ml of trypsinneutralizing solution (TNS) is added to stop the enzymatic reaction. Anadditional 5 ml of HEPES is added, and the cells are enumerated using ahemocytometer. The cell suspension is centrifuged and adjusted to adensity of, in the case of human cells, approximately 1.75×10⁶ cells/mlusing EGM-2 without antibiotics, or in the case of porcine cells,approximately 1.50×10⁶ cells/ml using EBM-2 supplemented with 5% FBS and50 μg/ml gentamicin.

Biocompatible Matrix. According to the present invention, theimplantable material comprises a biocompatible matrix. The matrix ispermissive for cell growth, and cell anchoring to and/or embeddingwithin the matrix. A particularly preferred matrix is one characterizedby a three-dimensional configuration such that anchored and/or embeddedcells can create and occupy a multi-dimensional habitat. Porous matricesare preferred. The matrix can be a solid or a non-solid. Certainnon-solid matrices are flowable and suitable for administration viainjection-type or infusion-type methods. In certain embodiments, thematrix is flexible and conformable. The matrix also can be in the formof a flexible planar form. The matrix also can be in the form of a gel,a foam, a suspension, a particle, a microcarrier, a microcapsule, or afibrous structure. In certain preferred embodiments, non-solid forms ofmatrix to which cells are anchored and/or in which cells are embeddedcan be injected or infused when administered.

One currently preferred matrix is Gelfoam® (Pfizer, New York, N.Y.), anabsorbable gelatin sponge (hereinafter “Gelfoam matrix”). Gelfoam matrixis a porous and flexible sponge-like matrix prepared from a speciallytreated, purified porcine dermal gelatin solution.

According to another embodiment, the biocompatible matrix material canbe a modified matrix material. Modifications to the matrix material canbe selected to optimize and/or to control function of the cells,including the cells' phenotype (e.g., the immunomodulatory phenotype) asdescribed elsewhere herein, when the cells are associated with thematrix. According to one embodiment, modifications to the matrixmaterial include coating the matrix with attachment factors or adhesionpeptides. Exemplary attachment factors include, for example,fibronectin, fibrin gel, and covalently attached cell adhesion ligands(including for example RGD) utilizing standard aqueous carbodiimidechemistry. Additional cell adhesion ligands include peptides having celladhesion recognition sequences, including but not limited to: RGDY,REDVY, GRGDF, GPDSGR, GRGDY and REDV.

According to another embodiment, the matrix is a matrix other thanGelfoam. Additional exemplary matrix materials include, for example,fibrin gel, alginate, polystyrene sodium sulfonate microcarriers,collagen coated dextran microcarriers, cellulose, PLA/PGA and pHEMA/MMAcopolymers (with polymer ratios ranging from 1-100% for each copolymer).According to a preferred embodiment, these additional matrices aremodified to include attachment factors, as recited and described above.

According to another embodiment, the biocompatible matrix material isphysically modified to improve cell attachment to the matrix. Accordingto one embodiment, the matrix is cross linked to enhance its mechanicalproperties and to improve its cell attachment and growth properties.According to a preferred embodiment, an alginate matrix is first crosslinked using calcium sulfate followed by a second cross linking stepusing calcium chloride and routine protocols.

According to yet another embodiment, the pore size of the biocompatiblematrix is modified. A currently preferred matrix pore size is about 25μm to about 100 μm; preferably about 25 μm to 50 μm; more preferablyabout 50 μm to 75 μm; even more preferably about 75 μm to 100 μm. Otherpreferred pore sizes include pore sizes below about 25 μm and aboveabout 100 μm. According to one embodiment, the pore size is modifiedusing a salt leaching technique. Sodium chloride is mixed in a solutionof the matrix material and a solvent, the solution is poured into amold, and the solvent is allowed to evaporate. The matrix/salt block isthen immersed in water and the salt leached out leaving a porousstructure. The solvent is chosen so that the matrix is in the solutionbut the salt is not. One exemplary solution includes PLA and methylenechloride.

According to an alternative embodiment, carbon dioxide gas bubbles areincorporated into a non-solid form of the matrix and then stabilizedwith an appropriate surfactant. The gas bubbles are subsequently removedusing a vacuum, leaving a porous structure.

According to another embodiment, a freeze-drying technique is employedto control the pore size of the matrix, using the freezing rate of theice microparticles to form pores of different sizes. For example, agelatin solution of about 0.1-2% porcine or bovine gelatin can be pouredinto a mold or dish and pre-frozen at a variety of differenttemperatures and then lyophilized for a period of time. The material canthen be cross-linked by using, preferably, ultraviolet light (254 nm) orby adding gluteraldehyde (formaldehyde). Variations in pre-freezingtemperature (for example −20° C., −80° C. or −180° C.), lyophilizingtemperature (freeze dry at about −50° C.), and gelatin concentration(0.1% to 2.0%; pore size is generally inversely proportional to theconcentration of gelatin in the solution) can all affect the resultingpore size of the matrix material and can be modified to create apreferred material. The skilled artisan will appreciate that a suitablepore size is that which promotes and sustains optimal cell populationshaving the phenotypes described elsewhere herein.

Cell Seeding of Biocompatible Matrix. The following is a description ofone exemplary configuration of a biocompatible matrix. As statedelsewhere, preferred matrices are solid or non-solid, and can beformulated for implantation, injection or infusion.

Pre-cut pieces of a suitable biocompatible matrix or an aliquot ofsuitable biocompatible flowable matrix are re-hydrated by the additionof EGM-2 without antibiotics at approximately 37° C. and 5% CO₂/95% airfor 12 to 24 hours. The implantable material is then removed from theirre-hydration containers and placed in individual tissue culture dishes.Biocompatible matrix is seeded at a preferred density of approximately1.5-2.0×10⁵ cells (1.25-1.66×10⁵ cells/cm³ of matrix) and placed in anincubator maintained at approximately 37° C. and 5% CO₂/95% air, 90%humidity for 3-4 hours to facilitate cell attachment. The seeded matrixis then placed into individual containers (Evergreen, Los Angeles,Calif.) tubes, each fitted with a cap containing a 0.2 μm filter withEGM-2 and incubated at approximately 37° C. and 5% CO₂/95% air. Themedia is changed every two to three days, thereafter, until the cellshave reached confluence. The cells in one preferred embodiment arepreferably passage 6, but cells of fewer or more passages can be used.

Cell Growth. A sample of implantable material is removed on or arounddays 3 or 4, 6 or 7, 9 or 10, and 12 or 13, the cells are counted andassessed for viability, and a growth curve is constructed and evaluatedin order to assess the growth characteristics and to determine whetherconfluence, near-confluence or post-confluence has been achieved.Generally, one of ordinary skill will appreciate the indicia ofacceptable cell growth at early, mid- and late time points, such asobservation of an exponential increase in cell number at early timepoints (for example, between about days 2-6 when using porcine aorticendothelial cells), followed by a near confluent phase (for example,between about days 6-8), followed by a plateau in cell number once thecells have reached confluence (for example, between about days 8-10) andmaintenance of the cell number when the cells are post-confluent (forexample, between about days 10-14).

Cell counts are achieved by complete digestion of the aliquot ofimplantable material with a solution of 0.5 mg/ml collagenase in aHEPES/Ca⁺⁺ solution. After measuring the volume of the digestedimplantable material, a known volume of the cell suspension is dilutedwith 0.4% trypan blue (4:1 cells to trypan blue) and viability assessedby trypan blue exclusion. Viable, non-viable and total cells areenumerated using a hemocytometer. Growth curves are constructed byplotting the number of viable cells versus the number of days inculture.

For purposes of the present invention, confluence is defined as thepresence of at least about 4×10⁵ cells/cm³ when in an exemplary flexibleplanar form of the implantable material (1.0×4.0×0.3 cm), and preferablyabout 7×10⁵ to 1×10⁶ total cells per aliquot (50-70 mg) when in aninjectable or infusible composition. For both, cell viability is atleast about 90% preferably but no less than 80%.

An exemplary embodiment of the present invention comprises abiocompatible matrix and cells suitable for use with any one of thevarious clinical indications or treatment paradigms described herein.Specifically, in one preferred embodiment, the implantable materialcomprises a biocompatible matrix and endothelial cells, endothelial-likecells, or analogs of either of the foregoing. In one currently preferredembodiment, the implantable material is in a flexible planar form andcomprises endothelial cells, preferably vascular endothelial cells suchas but not limited to human aortic endothelial cells and thebiocompatible matrix Gelfoam® gelatin sponge (Pfizer, New York, N.Y.,hereinafter “Gelfoam matrix”).

Implantable material of the present invention comprises cells anchoredto and/or embedded within a biocompatible matrix. Anchored to and/orembedded within means securely attached via cell to cell and/or cell tomatrix interactions such that the cells withstand the rigors of thepreparatory manipulations disclosed herein. As explained elsewhereherein, an operative embodiment of implantable material comprises anear-confluent, confluent or post-confluent cell population having apreferred phenotype. It is understood that embodiments of implantablematerial likely shed cells during preparatory manipulations and/or thatcertain cells are not as securely attached as are other cells. All thatis required is that implantable material comprise cells that meet thefunctional or phenotypical criteria set forth elsewhere herein.

The implantable material of the present invention was developed on theprincipals of tissue engineering and represents a novel approach toaddressing the herein-described clinical needs. The implantable materialof the present invention is unique in that the viable cells anchored toand/or embedded within the biocompatible matrix are able to supply tothe site of administration multiple cell-based products in physiologicalproportions under physiological feed-back control. As describedelsewhere herein, the cells suitable for use with the implantablematerial are endothelial, endothelial-like cells, or analogs of each ofthe foregoing. Local delivery of multiple compounds by these cells andphysiologically-dynamic dosing provide more effective regulation of theprocesses responsible for modulating an immune response. The implantablematerial of the present invention can provide an environment whichmimics supportive physiology and is conducive to modulation of an immuneresponse.

Evaluation of Functionality. For purposes of the invention describedherein, the implantable material is tested for indicia of functionalityprior to delivery to a recipient. For example, as one determination ofsuitability, conditioned media are collected during the culture periodto ascertain levels of heparan sulfate or transforming growth factor-β1(TGF-β1) or basic fibroblast growth factor (b-FGF) or nitric oxide whichare produced by the cultured endothelial cells. In certain preferredembodiments, the implantable material can be used for the purposesdescribed herein when total cell number is at least about 1, preferablyabout 2, more preferably at least about 4×10⁵ cells/cm³ of flexibleplanar form; percentage of viable cells is at least about 80-90%,preferably ≧90%, most preferably at least about 90%; heparan sulfate inconditioned media is at least about 0.1-0.5 preferably at least about0.23 microg/mL/day. If other indicia are desired, then TGF-β1 inconditioned media is at least about 200-300, preferably at least about300 picog/ml/day; b-FGF in conditioned media is below about 200picog/ml, preferably no more than about 400 picog/ml.

Heparan sulfate levels can be quantitated using a routinedimethylmethylene blue-chondroitinase ABC digestion spectrophotometricassay. Total sulfated glycosaminoglycan (GAG) levels are determinedusing a dimethylmethylene blue (DMB) dye binding assay in which unknownsamples are compared to a standard curve generated using knownquantities of purified chondroitin sulfate diluted in collection media.Additional samples of conditioned medium are mixed with chondroitinaseABC to digest chondroitin and dermatan sulfates prior to the addition ofthe DMB color reagent. All absorbances are determined at the maximumwavelength absorbance of the DMB dye mixed with the GAG standard,generally around 515-525 nm. The concentration of heparan sulfate perday is calculated by subtracting the concentration of chondroitin anddermatan sulfate from the total sulfated glycosaminoglycan concentrationin conditioned medium samples. Chondroitinase ABC activity is confirmedby digesting a sample of purified chondroitin sulfate. Conditionedmedium samples are corrected appropriately if less than 100% of thepurified chondroitin sulfate is digested. Heparan sulfate levels mayalso be quantitated using an ELISA assay employing monoclonalantibodies.

If desired, TGF-β1 and b-FGF levels can be quantitated using an ELISAassay employing monoclonal or polyclonal antibodies, preferablypolyclonal. Control collection media can also be quantitated using anELISA assay and the samples corrected appropriately for TGF-β1 and b-FGFlevels present in control media. Nitric oxide (NO) levels can bequantitated using a standard Griess Reaction assay. The transient andvolatile nature of nitric oxide makes it unsuitable for most detectionmethods. However, two stable breakdown products of nitric oxide, nitrate(NO₃) and nitrite (NO₂), can be detected using routine photometricmethods. The Griess Reaction assay enzymatically converts nitrate tonitrite in the presence of nitrate reductase. Nitrite is detectedcalorimetrically as a colored azo dye product, absorbing visible lightin the range of about 540 nm. The level of nitric oxide present in thesystem is determined by converting all nitrate into nitrite, determiningthe total concentration of nitrite in the unknown samples, and thencomparing the resulting concentration of nitrite to a standard curvegenerated using known quantities of nitrate converted to nitrite.

Also, any one or more of the foregoing assays can be used alone or incombination as a screening assay for identifying a cell as suitable foruse with the implantable material of the present invention.

While the earlier-described preferred immunomodulatory phenotype can beassessed using one or more of the optional quantitative heparin sulfate,TGF-β1, NO and/or b-FGF functional assays described above, implantablematerial can be evaluated for the presence of one or more preferredimmunomodulatory phenotypes as follows. For purposes of the presentinvention, one such preferred, readily identifiable phenotype typical ofcells of the present invention is an altered immunogenic phenotype asmeasured by the in vitro assays described below. Another readilyidentifiable phenotype typical of cells of the present invention is anability to block or interfere with dendritic cell maturation as measuredby the in vitro assays described below. Each phenotype is referred toherein as an immunomodulatory phenotype and cells exhibiting such aphenotype have immunomodulatory functionality.

Evaluation of Immunomodulatory Functionality: For purposes of theinvention described herein, the immunomodulatory functionality ofimplantable material can be tested as follows. For example, samples ofthe implantable material are evaluated to ascertain their ability toreduce expression of MHC class II molecules, to reduce expression ofco-stimulatory molecules, to inhibit the maturation of co-cultureddendritic cells, and to reduce the proliferation of T cells. In certainpreferred embodiments, the implantable material can be used for thepurposes described herein when the material is able to reduce expressionof MHC class II molecules by at least about 25-80%, preferably 50-80%,most preferably at least about 80%; to reduce expression ofco-stimulatory molecules by at least about 25-80%, preferably 50-80%,most preferably at least about 80%; inhibit maturation of co-cultureddendritic cells by at least about 25-95%, preferably 50-95%, mostpreferably at least about 95%; and/or reduce proliferation oflymphocytes by at least about 25-90%, preferably 50-90%, most preferablyat least about 90%.

Levels of expression of MHC class II molecules and co-stimulatorymolecules can be quantitated using routine flow cytometry andFACS-analysis, described in detail below. Proliferation of lymphocytescan be quantitated can be quantitated by in-vitro coculturing³[H]-thymidine-labeled CD3+-lymphocytes with the implantable compositionvia scintillation-counting as described below in detail. Inhibition ofdendritic cell maturation can be quantitated by either co-culturing theimplantable material with dendritic cells and evaluating surfaceexpression of various markers on the dendritic cells by flow cytometryand FACS analysis, or by measuring dendritic cell uptake ofFITC-conjugated dextran by flow cytometry. Each of these methods isdescribed in detail below.

Also, any one or more of the foregoing assays can be used alone or incombination as a screening assay for identifying a cell as suitable foruse with the implantable material of the present invention.

Methods of Use and Clinical Indications: This invention is directedgenerally to materials and methods for modulating an immunologicallyadverse response, including an inflammatory reaction, to an exogenousimmunogen or stimulus as well as an endogenous immunogen or stimulus.The invention is also directed to a cell-, tissue-, or organ-associatedimmunogen. For example, the present invention can modulate an adverseimmune response to non-syngeneic or syngeneic cells, tissues or organsand/or ameliorate a pre-existing immune condition such as but notlimited to an autoimmune condition. This discussion of implantablematerials and methods of use for suitable clinical indications will makereference to the following terms and concepts.

An early phase immune response depends on innate immunity. During aninnate immune response, a variety of innate immune mechanisms recognizeand respond to the presence of immunogen. Innate immunity is present inall individuals at all times and principally discriminates between self,altered self and non-self. For example, a type of innate immune cell isthe Natural Killer (NK) cell, the dendritic cell and the monocyte. Theinnate immune response is followed by an adaptive immune response,mediated by clonal selection of specific lymphocytes and resulting in amore tailored and long-lasting immune response against the recognizedantigen.

The adaptive immune response, or adaptive immunity, is the response ofantigen-specific lymphocytes to antigen, including the development ofimmunological memory. Adaptive immune responses are generated by clonalselection of lymphocytes. Adaptive immune responses are distinct frominnate and non-adaptive phases of immunity, which are not mediated byclonal selection of antigen-specific lymphocytes. The adaptive immuneresponse includes both cell-mediated immunity and humoral immunity. Forexample, an adaptive immune cell is a B-cell or T-cell lymphocyte.

One of the hallmarks of an adaptive immune response is establishment ofimmunological memory. Immunological memory is the ability of the immunesystem to respond more rapidly and effectively to immunogens beenencountered previously, and reflects the pre-existence of a clonallyexpanded population of antigen-specific lymphocytes.

Protective immunity can be either cell-mediated immunity or humoralimmunity. Humoral immunity is specific immunity mediated by antibodiesmade in a humoral immune response. Cell-mediated immunity describes anyadaptive immune response in which antigen specific T cells play a mainrole.

Autoimmune diseases are mediated by sustained adaptive immune responsesspecific for self antigens. Tissue injury results because the antigen isan intrinsic component of the body and consequently effector mechanismsof the immune system are directed at self tissues. Also, since theoffending autoantigen can not be removed from the body, the immuneresponse persists, and there is a constant supply of new autoantigen,which amplifies the response.

Although some syngeneic grafts or transplants may be accepted long-term,even syngeneic grafts can be problematic for a recipient. In fact, evenwhen autologous cells are harvested, manipulated ex vivo and returned tothe original donor, non-acceptance may occur to some extent. Typically,grafts differing at the MHC or at other genetic loci are rejected in theshort term by a recipient T-cell response. When donor and recipientdiffer at the MHC, for example, the immune response is directed at thenon-self MHC molecule or other surface molecules expressed by the graft.Acceptance or rejection of a graft or transplant invokes immune eventssuch as antigen recognition, T-cell activation, T-helper cellrecruitment and ultimately graft destruction.

An inflammatory reaction is initiated by a local immune response. Acuteinflammation is an early transient episode, while chronic inflammationpersists such as during autoimmune responses. Inflammation reflects theeffects of cytokines on local blood vessels. Cytokines have importanteffects on the adherent properties of the blood vessel endothelium,causing circulating leukocytes to stick to the endothelial cells of theblood vessel wall and migrate through the wall. Later-stage inflammatoryresponses also involve lymphocytes of the adaptive immune response whichhave been activated by immunogen.

Exemplary methods of treatment and clinical indications are discussedbelow. This is not intended to be an exhaustive discussion. The presentinvention contemplates any clinical indication suitable for treatmentwith the present invention, including any clinical indication typifiedby or otherwise associated with an immunological event having adverseclinical consequences for a patient.

Syngeneic and Non-syngeneic Transplants: The present invention can beused to reduce or diminish a transplant recipient's adverse response toa cell, tissue and/or organ transplant, whether it be a syngeneic or anon-syngeneic transplant. The present invention can also be used tostabilize or maintain a transplant recipient's acceptance of a cell,tissue or organ transplant, whether it be a syngeneic or a non-syngeneictransplant. As taught herein, modulation of an adverse immune responseoccurs when implantable material is used as a pre-transplant treatment,coincident treatment or post-transplant treatment. For example, it iscontemplated that a pre-treatment can acclimate a recipient's immunesystem which facilitates later acceptance of the transplant. Similarly,coincident treatment can shorten the time course of physiological eventswhich ultimately result in acceptance and ameliorate any adverseimmunological events provoked by the transplant. Post-transplanttreatments, whether single or multiple, can perpetuate a state ofacceptance and keep adverse immunological events in check if/when suchevents occur. Clinically, typical indications suitable for treatmentwith the implantable material of present invention include, but are notlimited to, allorejection, xenorejection, ischemia-reperfusion injuryassociated with transplanted tissues or organs, and repetitive treatmentcourses. Repetitive treatment courses include, for example, recurrentatherosclerosis at different vessel sites requiring repetitiveintervention and repetitive replenishing injections of pancreas isletcells. For purposes of the present invention, blood is a type of tissueand blood transfusion recipients can benefit from treatment with thepresent invention for all the foregoing reasons. Similarly,immunological-based diseases associated with cell, tissue and/or organtransplants benefit from the treatment paradigms set forth above.

Complement dependent Cytotoxicity: In addition to reducing, modulatingor eliminating the innate immune response and/or the adaptive immuneresponse, as outlined above, the implantable material of the presentinvention can also reduce, modulate or eliminate the severity of thecomplement cascade and the inflammatory side effects of complementactivation. For example, attenuation of the complement cascade using theimplantable material or the present invention reduces complementmediated cell lysis of a transplanted tissue or organ, therebyameliorating transplant dysfunction and extending the duration ofsuccessful treatment.

Interventional Therapies: As taught herein, the present invention canmodulate the severity or robustness of an already-existing immuneresponse as well as a future response provoked by subsequent earlierexposure(s) to an immunogen. Under such circumstances, implantablematerial can intervene by blocking escalation of an adverse immuneresponse or diminishing onset of hypersensitivity, respectively.Suppression of a memory response can avoid further physiological insultwhich can jeopardize a patient's organ health, for example. In the caseof an already-existing condition, such as an auto-immune condition, thepresent invention can quell the devastating effects of unabatedimmunological assaults on a patient's tissues or organs. In essence,such patients are continuously exposed to offending immunogen and theirimmune response escalates out-of-control resulting in serious, oftenfatal, disease sequalae.

While an auto-immune condition can be likened to serial challenges withan offending immunogen, other clinical indications can be consideredsimilarly. For example, as suggested above, a recipient of a syngeneicor non-syngeneic transplant is subject to serial challenges.Replenishment of a transplant, such as kidney islet cells whichdeteriorate over time, constitutes a serial challenge. Secondaryinfarctions or secondary vascular injuries can be considered serialchallenges. Another example is a disease such as but not limited tovasculitis. Any of the foregoing can be effectively managed using thematerials and methods of the present invention.

Supplanting Immunosuppressive Agents: As explained elsewhere herein, itis contemplated that administration of the implantable material of thepresent invention inhibits sufficiently at least T cell activation suchthat the need to administer harmful immunosuppressive agents iseliminated or significantly reduced. However, it is also contemplatedthat a certain class of patients, such as a patient pre-disposed tohighly exacerbated immune responses, can be treated with bothimplantable material and an immunosuppressive agent. The implantablematerial of the present invention, when administered prior to orcoincident with transplantation of syngeneic or non-syngeneic tissue,can permit reduced dosages of immunosuppressive agent, if one isnecessary, to manage a potential graft rejection response.

Potent immunosuppressive agents, for example, cyclosporin A, tacrolimus(FK-506), sirolimus (rapamicin), mycophenolate mofetil, leflunomide,glucocorticoids, cytostatics, azathioprine, and prednisone, areadministered to a transplant recipient to inhibit T cell activation andincrease the probability of graft survival. However, administration ofpotent immunosuppressive agents increases the risk of cancer andinfection and contributes to the risk of other side effects includinghypertension, dyslipidemia, hyperglycemia, peptic ulcers, and liver andkidney injuries. The present invention can permit more prudent and lessrisky dosing regimens of such agents. Additionally, immunosuppressantswhich are typically administered to an organ recipient can beadministered prior to, coincident with and/or subsequent toadministration of the implantable material of the present invention. Forexample, implantable materials can amplify the beneficial effects ofimmunosuppressants while minimizing the risks of such agents inrecipients whose immune system is overstimulated or over-sensitized,perhaps reducing the time in which immunomodulation is actuallyachieved. It is further contemplated that dosages of immunosuppressants,in certain embodiments, are less than those typically administered inthe absence of implantable material, thereby exposing a recipient toless toxic doses of immunosuppressants.

Altering the Time Course of an Immune Response: In a preferredembodiment of the invention, matrix-anchored and/or embedded endothelialcells are administered to diminish or delay an immune or inflammatoryresponse. It is not necessary that the implantable material completelyeliminate an immune or inflammatory response to be considered effective.Rather, the material need only alter the time course of a response, suchas by reducing the duration of an immune or inflammatory response or byreducing an acute inflammatory response to a chronic inflammatoryresponse. Delaying an immune or inflammatory response allows acoincident or later administered therapy to effectively treat arecipient in the absence of an immune or inflammatory response and/or toincrease the duration of transplant acceptance. Thus any delay orreduction in the severity of an adverse immune response is beneficialclinically to a patient.

Furthermore, the implantable material of the present invention can alsobe used to manage or reduce an immune response and inflammatory reactionassociated with any exogenous foreign body or foreign materialintroduced to a patient, or any form of exogenous stimulus. The presentinvention contemplates exogenous immunogens which arenaturally-occurring. The present invention also contemplates exogenousimmunogens, including but not limited to pharmaceutical agents, toxins,surgical implants, infectious agents and chemicals. For purposes of thepresent invention, an exogenous immunogen can be an exogenous stimulussuch as, but not limited to, environmental stress, injury, exposure orany stimulus which provokes an adverse immune response or inflammatoryreaction.

For example, synthetic graft materials, such as a synthetic PTFE®arteriovenous graft, or other synthetic surgical materials or prostheticdevices, can induce a foreign body reaction in the host. This type ofimmune or inflammatory response can also be reduced or eliminated byadministering the implantable material of the present invention to thepatient prior to or at the time of implanting the synthetic material.Administration subsequent to implantation is also effective. Reducingany foreign body reaction in the host improves the overall functionand/or outcome of the treatment.

General Considerations. In certain embodiments of the invention,additional therapeutic agents are administered prior to, coincident withand/or following administration of the implantable material. Forexample, cytokines or growth factors can also be incorporated into theimplantable material, depending on the clinical indication necessitatingthe implant, including agents which can mute an immune-related humoralor cellular event, or tissue-associated biochemical cascade. Other typesof therapeutic agents include those which can promote the longevity ofcells anchored to and/or embedded within the implantable material and/oragents which can delay the bioerosion of an erodible biocompatiblematrix post implantation. Any of the foregoing can be administeredlocally or systemically; if locally, certain agents can be containedwithin the implantable material or contributed by the cells per se.

Administration Considerations. As contemplated herein, the implantablematerial of the present invention can be delivered to or situated at anycompatible anatomical site provided that conditions at the site do notcause mechanical-type or physical-type disruption or untimelydisintegration of the implantable material, or otherwise compromise thephysical integrity or the functionality of the implantable material. Forexample, the present invention can be situated subcutaneously,perivascularly, or intraperitoneally. One preferred site is a skinpouch. Other preferred sites can be perivascular or non-perivascular.The implantable material can be situated adjacent to or in contact withan organ or a tubular anatomical structure which can be a vascular ornon-vascular structure. The present invention can be delivered to anycompatible site for purposes of either systemic modulation of a humoralor cellular immune response, or for purposes of local modulation of aninflammatory reaction, or both. Certain preferred embodiments ofimplantable material can reside at an implantation site for at leastabout 56-84 days, preferably about at least 7 days, more preferablyabout at least 14 days, even more preferably about at least 28 days, andmost preferably more than about 28 days before it bioerodes.

When ready for delivery to a recipient, the implantable material when inan exemplary flexible planar form, is a 1×4×0.3 cm (1.2 cm³) sterilepiece with preferably approximately 5-8×10⁵ preferably at least about4×10⁵ cells/cm³ and at least about 90% viable cells, for example, humanaortic endothelial cells derived from a single cadaver donor source, percubic centimeter in approximately 45-60 ml, preferably about 50 ml,endothelial growth medium (for example, endothelial growth medium(EGM-2) containing no phenol red and no antibiotics. When porcine aorticendothelial cells are used, the growth medium is also EBM-2 containingno phenol red, but supplemented with 5% FBS and 50 μg/ml gentamicin.

In certain embodiments contemplated herein, the implantable material ofthe present invention is a flowable composition comprising a particulatebiocompatible matrix which can be in the form of a gel, a foam, asuspension, a particle, a microcarrier, a microcapsule, or otherflowable material. Any non-solid flowable composition for use with aninjection-type or infusion-type delivery device is contemplated herein.In certain embodiments, the flowable composition is preferably ashape-retaining composition. An implantable material comprising cellsin, on or within a flowable-type particulate matrix as contemplatedherein can be formulated for use with any injection-type delivery deviceranging in internal diameter from about 22 gauge to about 26 gauge andcapable of delivering about 50 mg of flowable composition comprisingparticulate material containing preferably about 1 million cells inabout 1 to about 3 ml.

According to a currently preferred embodiment, the flowable compositioncomprises a biocompatible particulate matrix such as Gelfoam® particles,Gelfoam® powder, or pulverized Gelfoam® (Pfizer Inc., New York, N.Y.)(hereinafter “Gelfoam particles”), a product derived from porcine dermalgelatin. According to another embodiment, the particulate matrix isCytodex-3 (Amersham Biosciences, Piscataway, N.J.) microcarriers,comprised of denatured collagen coupled to a matrix of cross-linkeddextran.

Endovascular Administration. The flowable composition can also beadministered via an intraluminal or endovascular route even though thefinal deposition site is not intraluminal. For example, the compositioncan be delivered by any device able to be inserted within the bloodvessel. Endoscopic guidance systems may be used to locate the deliverydevice at the site of administration, including, for example,intravascular ultrasound (IVUS), color Doppler ultrasound, duplexultrasound, other routine ultrasound, angiography, magnetic resonanceangiography (MRA), magnetic resonance imaging (MRI), CT scanning,fluoroscopy to identify the location of a stent and/or other endoscopicguidance systems known in the field. Additionally, the site ofadministration may be located using tactile palpation.

In one instance, the intraluminal delivery device is equipped with atraversing or penetrating device which penetrates the luminal wall of ablood vessel to reach a non-luminal surface of a blood vessel. Theflowable composition is then deposited on the non-luminal surface. It iscontemplated herein that a non-luminal, also termed an extraluminal,surface can include any site exterior to a blood vessel or anyperivascular surface of a vessel, or can be within the adventitia,media, or intima of a blood vessel, for example. For purposes of thisinvention, non-luminal or extraluminal means any surface except aninterior surface of the lumen. It is also contemplated that depositionwithin the perivascular space can be accomplished via an intraluminaldelivery device and does not require contact with the extraluminalsurface of the traversed vessel.

The penetrating devices contemplated herein can permit, for example, asingle point of delivery or a plurality of delivery points arranged in adesired geometric configuration to accomplish delivery of the flowablecomposition to a non-luminal surface of a blood vessel withoutdisrupting an injured or diseased target site. A plurality of deliverypoints can be arranged, for example, in a circle, a bulls-eye, or alinear array arrangement to name but a few. The penetrating device canalso be in the form of a stent perforator, such as but not limited to, aballoon stent including a plurality of delivery points.

Percutaneous Administration. Flowable composition can be delivered via apercutaneous route using a needle, catheter or other suitable deliverydevice. The flowable composition can be delivered percutaneouslycoincident with use of a guidance method to facilitate delivery to thesite in need of treatment. The guidance step is optional. Endoscopicguidance systems can be used to locate a site of extraluminaladministration, for example, including intravascular ultrasound (IVUS),color Doppler ultrasound, duplex ultrasound, other routine ultrasound,angiography, magnetic resonance angiography (MRA), magnetic resonanceimaging (MRI), CT scanning fluoroscopy. Additionally, the site ofadministration can be located using tactile palpation. Upon entry intothe perivascular or peritoneal space, for example, the clinician candeposit the flowable composition on any non-luminal surface or at anynon-luminal site. The guiding or identifying step is optionallyperformed and not required to practice the methods of the presentinvention. In another embodiment, the implantable flowable compositionis delivered locally to a surgically-exposed extraluminal site.

Also contemplated is administration by infusion. Infusion can beaccomplished as a bolus-type dose or a slower-type, gradual dose. Theskilled clinician will recognize the advantages of each and willrecognize the circumstances in which to employ one or the other modes ofadministration. All that is required is routine clinical infusionapparatus.

Experimental Materials and Procedures

Material Preparation and Evaluation. As described in greater detailelsewhere herein, porcine aortic endothelial cells and human aorticendothelial cells were individually isolated and cultured. The culturedcells were then seeded on a three-dimensional biocompatible matrix, suchas Gelfoam, and incubated until the cells reached confluence. Thefunctionality of the endothelial cells anchored to and/or embeddedwithin the matrix was evaluated according to the previously discussedprotocols.

Endothelial cell-induced immune reaction in rats. Fifty-fourSprague-Dawley rats received 5×10⁵ porcine aortic endothelial celltransplants in the subcutaneous dorsal space as Gelfoam-embedded cells,saline-suspended cell pellets, or as pellets adjacent to empty Gelfoam.After dorsal incision, a small subcutaneous cavity was built in blunttechnique and Gelfoam-embedded cells carefully inserted or cellsinjected. Empty control Gelfoam matrices were incubated in complete DMEMprior to implantation. Sera were collected serially from 0 to 56 days,aliquoted and stored at −70° C.

Endothelial cell-induced immune reaction in mice. Thirty-six B6-micereceived 5×10⁵ porcine aortic endothelial cell implants in thesubcutaneous dorsal space as Gelfoam-embedded cells, saline-suspendedcell pellets, or as pellets adjacent to empty Gelfoam. Empty controlGelfoam matrices were incubated in complete DMEM prior to implantation.To evaluate the impact of matrix-embedding on immunological memory thesame groups of mice were rechallenged with the identical treatment onday 100. Sera were collected serially from 0 to 90 days after eachimplantation-procedure, aliquoted and stored at −70° C. Four mice ofeach group were sacrificed on day 28 and day 128 respectively forsplenocyte isolation.

Endothelial cell-induced immune reaction in serially challenged mice.Porcine aortic endothelial cells (PAE) isolated from LargeWhite swineaorta were either seeded on Gelfoam as previously described or grown toconfluence on polystyrene plates. B6-mice received injections in thesubcutaneous dorsal space on days 0, 21, 35 of 5×10⁵ PAE (n=24,pre-sensitized mice) or saline (n=24, naïve mice). On day 42, 12 micefrom each group received 5×10⁵ matrix-embedded or free PAE. Host immunereactions and lytic damage of endothelial cells were studied for thefollowing 90 days. Sera were collected serially from days 42 to 132,aliquoted and stored at −70° C. Six mice of each group were sacrificedon day 70, the remaining on day 132 for splenocyte isolation.

Experiments Endothelial Cells Embedded in a Three-Dimensional MatrixGrow in a Three-Dimensional Pattern.

Scanning electron microscopy was performed to evaluate the growingpattern of endothelial cells grown within a biocompatible matrix.Implantable material comprising endothelial cell anchored to and/orembedded within a Gelfoam matrix were rinsed with PBS, divided into 0.5cm specimens, fixed with 3% glutaraldehyde (Sigma Chemicals; St. Louis,Mo.) 90 min, and transferred to distilled water. After incubation in 1%OsO4, specimens were rinsed with distilled water and dehydrated inserial solutions of ethanol (30, 50, 75, 80, 85, 90, 95, and 100%) at 15min intervals, and hexamethyldisilazane (Sigma) (50%, 100%) at 30 minintervals. Specimens were evaporated overnight in 100% HMDS andthereafter coated with gold in a plasma coater (Edwards Coating System,U.K.). Scanning electron microphotographs were obtained at 5-kVacceleration voltage (Stereoscan 240, Cambridge Instruments, U.K.).

Scanning electron microscopy revealed a 3-D growing pattern of porcineaortic endothelial cells along the interstices of the Gelfoam-matrix.Cell viability remained at 95% over the 2-week culture course.

Experimental data indicate that the in-vivo immunoacceptance ofGelfoam-embedded cells is an effect of the three-dimensional growingpattern of endothelial cells in the matrix rather than from the presenceof the biocompatible matrix alone. Typically, implanted cells orproteins combined within tissue-engineered biomaterials serve as asource of antigens immuno-stimulating. Yet, the Gelfoam matrix isimmunoneutral and itself has no immune protective effect since injectionof porcine aortic endothelial cells adjacent to Gelfoam matrix aloneevoked the same immune response as free injected endothelial cells. Thenature of endothelial cells contributes to this unique form ofimmunomodulation observed with matrix-embedded cell preparations. Inparticular, these cells have a sidedness: a basal surface that interactswith basement membrane and superior surface that interacts with flowingblood and cellular elements. Data suggest that endothelial cell functionis anchorage- and density-dependent. Systemic diseases likehypertension, alterations in lipid and glucose metabolism or exposure totoxins alter anchorage-dependent regulation and the amplitude and natureof immune responses against the endothelium and phenotypictransformation of intact endothelial cells from matrix-adherent to freecontributes to initiation of vascular disease.

Modulation of Surface Molecules Including Co-Stimulatory and AdhesionMolecules.

Expression levels of costimulatory and adhesion molecules on endothelialcells in vitro were quantified by flow cytometry. FITC- and PE-labeledantibodies were used and included mouse anti-porcine P-selectinantibody, mouse anti-porcine CD31 (clone LCI-4), anti-human CD54 (clone15.2), anti-human CD62E (clone 1.2B6), anti-human CD58 (clone 1C3),anti-human CD80 (clone BB1), anti-human CD86 (clone 2331), anti-human4-1BB-ligand (PE-labeled, clone C65-485), rat anti-mouse IgG₁ (cloneA85-1), and anti-mouse IgM (clone R6-60.2), rabbit anti-rat IgG, rabbitanti-human CD40, goat anti-rabbit IgG, mouse anti-human CD106 (clone1.G11B1), mouse anti-human HLA-DP,DQ,DR (clone CR3/43), mouse anti classI MHC (IgG_(2a)), rat anti-mouse IgG_(2a), mouse anti-human ox40-ligand,mouse anti-human Programmed Death Ligand 1 (PD-L1, clone MIH1),anti-human PD-L2 (clone MIH18), and anti-human inducible costimulatorligand (ICOS-ligand, clone MIH12).

Endothelial cell monolayers or endothelial cells embedded in Gelfoamwere harvested after culture in complete medium (CD31, CD58, PD-L2,ox40-ligand, MHC-I), stimulated with 100 U/ml TNF-α (CD54, CD80, CD86,CD106, E-selectin, P-selectin) or 200 U/ml TNF-α (ICOS-L) for 24 hours,10 μg/ml LPS for 24 hours (4-1BB-ligand), 1000 U/ml IFN-γ (MHC-II,CD40), or 100 U/ml IFN-γ and 25 ng/ml TNF-α (PD-L1) for 48 hours. Mediawere aspirated and cells were washed with PBS. Monolayers incubated in1.0 mM PBS/EDTA for 5 min, and disrupted by gentle shaking. Gelfoam weredigested with collagenase type I, shown to have no effect on expressionof surface molecules. Cell-suspensions were washed and 3×10⁵ cells wereresuspended in FACS buffer (PBS containing 0.1% BSA and 0.1% sodiumazide, Sigma Chemicals; St. Louis, Mo.). Endothelial cells wereincubated with primary antibodies for 30 min at 4° C. If necessary,cells were resuspended in FACS buffer and stained with a secondaryantibody for 30 min at 4° C. Cells were then washed, fixed in 1%paraformaldehyde, and 10⁴ cells were analyzed by flow cytometry using aFACScalibur instrument and CellQuest software (Becton Dickinson, SanDiego, Calif.).

Embedding porcine aortic endothelial cells in a three-dimensionalbiocompatible matrix altered the expression of surface molecules.Constitutive expression of CD58 was significantly reduced in porcineaortic endothelial cells embedded in Gelfoam compared to CD58 expressionof porcine aortic endothelial cells grown on tissue culture polystyreneplates (−60.4%, p<0.002). There was also a significant reduction inupregulation of costimulatory and adhesion molecules, and MHC class IIon matrix-embedded porcine aortic endothelial cells compared to porcineaortic endothelial cells grown on polystyrene plates under FACS-analysis(CD80: —64.9%, p<0.002; CD86: —65.4%, p<0.001; CD40: —53.8%, p<0.005;ICAM-1: —68.7%, p<0.001; VCAM-1: —53.9%, p<0.005; E-selectin: —71.8%,p<0.0005; P-selectin: —79.9%, p<0.0002; MHC II: —78.3%, p<0.0002). Therewere no significant differences in surface expression of MHC class I andCD31.

Similarly, embedding human aortic endothelial cells in athree-dimensional biocompatible matrix altered the expression of surfacemolecules. Human aortic endothelial cells grown in a 3D matrix exhibiteda significantly reduced expression profile of CD58 and showed asignificant lack in upregulation of costimulatory and adhesionmolecules. However, there were no significant differences in ICAM-1,E-selectin, MHC I, and CD31 expression levels between human aorticendothelial cells embedded in Gelfoam and human aortic endothelial cellsgrown on tissue culture polystyrene plates. Furthermore, there were nosignificant differences in constitutive expression of PD-L2 (100%,p=0.73) and in upregulation of PD-L1 (86%, p=0.09).

Thus, embedding endothelial cells in a three-dimensional biocompatiblematrix reduces costimulatory and adhesion molecules. Matrix embeddedporcine aortic endothelial cells and human aortic endothelial cellsexhibited significantly lower expression levels of costimulatory andadhesion molecules on activated endothelial cells.

Expression of CD31, MHC-11, CD58, ICAM-1 and E-selectin was alsoanalyzed in the implants in vitro by confocal microscopy and in rats invivo by immunohistochemical analysis. Endothelial cells were seeded oncover slips or embedded in Gelfoam-matrices. After washing with PBS andfixation with 3% paraformaldehyde for 20 min (cover slips) or overnight(Gelfoam), endothelial cells were blocked with rat serum (BethylLaboratories, TX) for 30 min. Before staining with antibodies, Gelfoammatrices were cut into 2 mm thick slices. Endothelial cells were stainedwith the appropriate amount of antibodies for 1 (cover slips) or 2 hours(Gelfoam) and analyzed on a Zeiss LSM510 Laser scanning confocalmicroscope. Staining intensity was quantified with ImageJ software(National Institute of Health, Bethesda, Md.) and normalized againstCD31 expression.

Confocal microscopy revealed reduced expression-levels of CD58, ICAM-1,E-selectin, and MHC-II on matrix embedded porcine aortic endothelialcells whereas CD31 expression remained unchanged (p<0.02).Cell-substrate anchoring had no effect on MHC-I expression but markedlymuted the expected upregulation of MHC-II molecules. Porcine aorticendothelial cells embedded in Gelfoam evoked only a modest proliferationof xenogeneic CD4⁺ T cells in-vitro similar to the response seen withblockade of MHC-II binding in free porcine aortic endothelial cells.

Modulation of the Immune Response In Vivo

Matrix embedded porcine aortic endothelial cells showed a lowerstimulation of the initial event in the recruitment of leukocytes whichinvolve P- and E-selectin, and of VCAM-1 which is closely associatedwith T cell recruitment at sites of immune inflammation. The full panelof general and species specific costimulatory molecules was downregulated by matrix embedding, including the first report of endothelialcell-expression and suppression of 4-1BB-ligand. At the same time,expression and upregulation of PD-L1 and PD-L2, members of the B7-familythat act as countervailing inhibitory molecules, remained intact aftermatrix embedding. These in-vitro findings translated into asignificantly muted immune reaction in rats after implantation ofmatrix-embedded porcine aortic endothelial cells.

The cellular response to implantation was also evaluatedimmunohistochemically in six rats from each group on day 28 postimplantation. Five-micrometer paraffin sections were cut and antigenretrieval performed by microwave heating for 10 minutes in a 0.01 mol/Lcitrate buffer, pH 6.0. Leukocytes, T and B lymphocytes were identifiedby an avidin-biotin peroxidase complex method. The primary antibodieswere mouse anti-rat CD45R0, to identify leukocytes (ResearchDiagnostics; 1:50 dilution), mouse anti-rat CD4, to identify CD4⁺-Tcells (Pharmingen; 1:10 dilution), and mouse anti-rat CD8, to identifyCD8⁺-T cells (Pharmingen; 1:50 dilution). Rat spleen was used as apositive control, and mouse IgG as negative controls. Primary antibodieswere applied for 1 hour at room temperature, and all sections werecounterstained with Mayer's hematoxylin solution (Sigma). Sixnonoverlapping fields (×600) were examined. The results for each groupwere averaged.

Embedding endothelial cells in a three-dimensional biocompatible matrix,as compared to injected free PAE or PAE injected adjacent to athree-dimensional biocompatible matrix, also reduced the immune responsein rats in vivo. Porcine aortic endothelial cells embedding in Gelfoamsignificantly reduced formation of porcine aortic endothelialcell-specific IgG in vivo. Serum cytokines (MCP-1, IL-6, TNF-α) rose,peaking five days after implantation, in rats receiving free porcineaortic endothelial cells and injections of porcine aortic endothelialcells adjacent to Gelfoam. In contrast, cytokine levels did not increaseabove background in animals with matrix-embedded porcine aorticendothelial cells.

Immunohistological studies revealed evidence of cellular infiltrationinto and around the implants/injection site at 28 days. After injectionof free porcine aortic endothelial cells and injection of porcine aorticendothelial cells adjacent to Gelfoam, T cells were abundant within theimplant/injection side, whereas large numbers of CD45R0 positiveleukocytes were also found at the periphery of the graft. In contrast,the tissue surrounding the implant and Gelfoam-porcine aorticendothelial cells itself were infiltrated with 4.5 fold fewer leukocytesand CD4⁺-T cells, and 3.3 fold fewer CD8⁺ T cells than the other cellimplantation groups.

Circulating rat immunoglobulins specific for the implanted porcineaortic endothelial cells were also measured by flow cytometry. 2×10⁵porcine aortic endothelial cells were detached from tissue culturepolystyrene plates with 0.25% trypsin/0.04% EDTA, pelleted, washed,resuspended in FACS buffer and incubated with serum from recipient ratsfor 30 min at 4° C. (diluted 1:10 in FACS buffer). After washing twicewith cold FACS buffer, cells were incubated with FITC-conjugatedanti-rat IgG. Following 30 min incubation at 4° C. in the dark, thesamples were again washed twice with cold FACS buffer, fixed in 1%paraformaldehyde, and 10⁴ cells were analyzed by flow cytometry using aFACScalibur instrument and CellQuest software. Rat IL-6 (R&D Systems,MN, detection limit 21 pg/ml), rat TNF-α (R&D Systems, detection limit<5 pg/ml), and rat MCP-1 (Amersham, detection limit <5 pg/ml)serum-concentrations were quantified by ELISA on days 0, 5, 12, and 28post implantation. Measurements were performed at the same time by thesame ELISA to avoid variations of assay conditions.

The levels of immunoglobulins specific for the implanted porcine aorticendothelial cells in serum of the experimental mice were also measuredby flow cytometry. 2×10⁵ porcine aortic endothelial cells, from the samestrain as the implanted cells, were detached from cell culture plateswith 0.25% trypsin/0.04% EDTA, pelleted, washed, and resuspended in FACSbuffer (PBS, 1% FCS, 0.1% sodium azide). These cells were then incubatedwith serum from recipient mice for 60 min at 4° C. (diluted 1:10 in FACSbuffer). After washing twice with FACS buffer, cells were incubated withFITC-conjugated rat anti-mouse IgG_(2a) (Southern biotechnology, AL),IgG₁ (clone A85-1), or IgM (clone R6-60.2, BD Pharmingen, CA)respectively. Following 30 min incubation at 4° C. in the dark, thesamples were again washed twice with cold FACS buffer, fixed in 0.25 ml1% paraformaldehyde, and 10 ⁴ cells were analyzed by flow cytometryusing a FACScalibur instrument and CellQuest software.

Embedded endothelial cells in a three-dimensional biocompatible matrix,as compared to injected free PAE and PAE injected adjacent to athree-dimensional biocompatible matrix, reduced the Th2-driven immuneresponse in mice in vivo. To characterize the magnitude and nature ofthe porcine aortic endothelial cell-specific antibody response, serumwas collected from mice after implantation of porcine aortic endothelialcells in the subcutaneous dorsal space as Gelfoam-embedded cells,saline-suspended cell pellets, or as pellets adjacent to empty Gelfoam.Post-implantation anti-porcine aortic endothelial cell IgG₁ and IgMlevels were similar and significantly higher in mice receiving porcineaortic endothelial cell pellets or porcine aortic endothelial cellpellets adjacent to empty Gelfoam compared to recipients of porcineaortic endothelial cells embedded in Gelfoam (FIGS. 1A and 1B). Therewas a transient and minor elevation in anti-porcine aortic endothelialcell IgG_(2a) 12 days after implantation (p<0.005) after implantation ofmatrix-embedded porcine aortic endothelial cell mice which was not seenin mice receiving pelleted porcine aortic endothelial cells or implantsof empty Gelfoam with injection of pelleted porcine aortic endothelialcells (FIG. 1C).

FIGS. 1A, 1B and 1C graphically depict circulating PAE-specific IgG inmice after subcutaneous injection of free PAE, of Gelfoam-grownendothelial cells, or after concomitant injection of PAE adjacent toGelfoam alone as determined via flow-cytometry. Graphic depiction ofresults from all mice (n=18 per group to day 28, n=12 per group day56-100 post-implantation) demonstrates a statistically significantdifference between the matrix-embedded and other forms of PAEimplantation for IgG₁ (FIG. 1A) and IgM (FIG. 1B). There was a transientand minor elevation in anti-PAE IgG_(2a) 12 days after implantation ofmatrix-embedded PAE (FIG. 1C).

Compared to unstimulated HAE grown on tissue culture plates,matrix-embedded HAE expressed significantly higher levels of theinhibitory signaling molecules suppressor-of-cytokine-signaling (SOCS)3(0.007±0.001 vs. 0.003±0.0003 RU, p<0.001). Hence stimulation with IFN-γresulted in significantly lower expression of MHC II on matrix-embeddedHAE (37±5 vs. 68±4%, p<0.001). Despite unchanged IFN-γ-receptorexpression levels (p=0.39) substrate adherence reduced IFN-γ-inducedphosphorylation of Janus kinase 1 and 2 andsignal-transducer-and-activator-of-transcription-1. This was followed bydiminished expression of interferon-regulatory factor-1, CIITA(0.01±0.004 vs. 0.03±0.004 RU, p<0.005), and HLA-DR (0.17±0.04 vs.0.27±0.05 RU, p<0.02) in matrix-embedded HAE. Reduced MHC II expressionon matrix-embedded HAE resulted in muted ability to induce proliferationof allogeneic T cells (4152±255 vs. 19619±327 cpm, p<0.001).

Interestingly, embedding endothelial cells in a three dimensional matrixnearly completely diminishes the observed Th2-driven immune response,mutes lytic activity and attenuates differentiation of naïve T cellsinto effector cells. In accordance with previous results, these datasuggest that Gelfoam embedding of cells provides immune protection byimmune activation at the T-cell level via reduced expression levels ofMHC class II molecules as well as costimulatory and adhesion molecules.

Modulation of Lymphocyte Proliferation and Lytic Activity.

Porcine aortic endothelial cells grown on polystyrene wells or embeddedin Gelfoam were seeded in 96 well plates at 5×10⁴ cells/well andstimulated with 40 ng/ml porcine INF-γ for 48 hours, followed bymitomycin C treatment (Sigma, 50 μg/ml for 30 min) to prevent backgroundproliferation. Human CD4⁺ lymphocytes were purified by negativeselection with a CD4⁺ T cell isolation kit II (Miltenyi Biotec, Germany)according to the manufacturer's instructions and added at 2×10⁵cells/well. In some experiments a murine antibody directed againstHLA-DP, DQ, DR blocked activation via MHC class II molecules.³[H]-thymidine incorporation was measured on day 6 by 16 h pulse (1μCi/ml, Amersham). Thymidine uptake of mitomycin-treated porcine aorticendothelial cells, medium or T cells alone was used as negativecontrols.

To evaluate lymphocyte lytic activity in mice in vivo, splenocyteisolation and evaluation was performed. Spleens of 4 mice from eachgroup were isolated aseptically in a laminar flow hood on day 28 afterporcine aortic endothelial cell-implantation. Organs were cut in severalpieces. Clumps were further dispersed by drawing and expelling thesuspension several times through a sterile syringe with a 19-Gaugeneedle. Afterwards, the suspension was expelled through a 200 μm meshnylon screen. Cells were washed twice with RPMI (containing 2 mML-glutamine, 0.1 M HEPES, 200 U/ml Penicillin G, 200 μg/ml streptomycinand 5% heat-inactivated calf serum, Life Technologies) and immediatelyused.

To further evaluate lymphocyte lytic activity in mice in vivo, aCalcein-AM release assay was performed. Porcine aortic endothelial cellsfrom the same strain of injected cells were resuspended in completemedium at a final concentration of 2×10⁴/well and incubated with 15 μMcalcein-AM (Molecular Probes) for 40 min at 37° C. with occasionalagitation. After two washes with complete medium, splenocytes aseffector cells were added at a final concentration of 5×10⁵/well.Spontaneous and maximum release were examined as controls in sixreplicate wells that contained only target cells in complete medium andsix wells with target cells in medium plus 2% Triton X-100 for the last20 minutes. After 3 hour incubation at 37° C./5% CO₂ samples weremeasured using a Fluoroskan Ascent FL dual-scanning microplateluminofluorimeter (Thermo Electron Corporation, TX). Data were expressedas arbitrary fluorescent units (AFU). Specific lysis was calculatedaccording to the formula [(test release−spontaneous release)/(maximumrelease−spontaneous release)]×100.

Embedding endothelial cells in a three-dimensional biocompatible matrixreduced lymphocyte proliferation. The proliferative response of isolatedhuman CD4⁺ T cells to untreated and INF-γ treated porcine aorticendothelial cells (40 ng/ml. 48 hours) grown in tissue culture plates orembedded in Gelfoam was assayed by thymidine incorporation. The strongCD4⁺ T cell proliferation noted after exposure to porcine aorticendothelial cells pretreated with INF-γ was nearly eliminated whenporcine aortic endothelial cells were matrix-embedded (17087.2±3749.75vs. 5367.8±1976.3 cpm, p<0.01). The presence of MHC II antibody blockedlymphocyte proliferation in response to INF-γ-treated porcine aorticendothelial cells by 65% to a level comparable to matrix embeddedporcine aortic endothelial cells. Mitomycin-treated porcine aorticendothelial cells did not show a significant proliferation after 6 dayculture (61±13 cpm) as well as culture of isolated CD4⁺ T cells alone(83±27 cpm).

Similarly, embedding endothelial cells in a three-dimensionalbiocompatible matrix, as compared to injected free PAE and PAE injectedadjacent to a three-dimensional biocompatible matrix, reduced lymphocytelytic activity in mice in vivo. Lymphocytes from mice spleens from thethree different treatment groups were isolated 28 days after porcineaortic endothelial cell implantation. Donor porcine aortic endothelialcells were labeled with Calcein-AM and endothelial cell-lysis wasmeasured by a calcein fluorescence release assay after coincubation withlymphocytes. Lymphocytes from mice after pure porcine aortic endothelialcell-injection (36.8±3.9%) and after concomitant porcine aorticendothelial cell-injection (33.9±4.7%) showed the highest lytic activityas compared to lymphocytes isolated from mice after implantation ofporcine aortic endothelial cell-Gelfoam constructs (22.4±4.2%, p<0.05;FIG. 2).

FIG. 2 graphically depicts splenocytes from mice receiving free PAE andshows significantly increased lytic activity when compared tomatrix-embedded PAE. Rechallenge of mice with free PAE significantlyincreased xenogeneic lytic activity of isolated splenocytes.

Modulation of Th2 Cytokine-Producing Cells and Cytokines.

Immunospot plates (Millipore, Bedford, Mass.) were coated with 5 μg/mlof anti-mouse interferon (IFN)-γ, anti-mouse interleukin (IL)-2,anti-mouse IL-4, or anti-mouse IL-10 mAb (all BD Pharmingen) in sterilePBS overnight. The plates were then blocked for two hours with completeRPMI-medium without phenol red, containing 10% heat-inactivated calfserum. Splenocytes (0.5×10⁶ in 100 μl complete RPMI-medium) and the samestrain of porcine aortic endothelial cells used for implantation(0.5×10⁶ in 100 μl complete RPMI-medium) were then placed in each welland cultured for 48 hours at 37° C. in 5% CO₂. After washing withdeionized water followed by washing with PBS containing 0.05% Tween(PBST), 2 μg/ml of biotinylated anti-mouse IFN-γ, anti-mouse IL-2,anti-mouse IL-4, or anti-mouse IL-10 mAb (all BD Pharmingen) were addedovernight respectively. The plates were then washed three times in PBST,followed by one hour of incubation with horseradishperoxidase-conjugated streptavidin (BD Pharmingen). After washing fourtimes with PBST followed by PBS, the plates were developed using3-amino-9-ethyl-carbazole (BD Pharmingen). The resulting spots werecounted on a computer-assisted enzyme-linked immunospot image analyzer(Cellular Technology Ltd., ORT). The number of spots in the wells withmedium, splenocytes or porcine aortic endothelial cells alone wassubtracted from xenoresponses to account for background in dataanalysis.

Embedding endothelial cells in a three-dimensional biocompatible matrix,as compared to injected free PAE and PAE injected adjacent to athree-dimensional biocompatible matrix, reduced Th2 cytokine-producingcells in mice in vivo. The frequency of Th1 cytokine (IFN-γ, IL-2) andTh2 cytokine (IL-4, IL-10)-producing cells was measured by ELISPOT assayin splenocytes recovered from animals after implantation of differentforms of porcine aortic endothelial cells. At day 28 postimplantation,the frequency of Th2 cytokine-producing cells was significantly lower insplenocytes isolated from mice receiving matrix embedded porcine aorticendothelial cells compared with those isolated from mice receiving freeporcine aortic endothelial cells or porcine aortic endothelial cellsadjacent to empty Gelfoam ((IL-4: p<0.0001, IL-10<0.001; FIG. 3A). Incontrast, there were no significant differences in the frequency of Th1cytokine-producing cells in splenocytes isolated from the three groups(FIG. 3B).

FIG. 3A graphically depicts the frequencies of xenoantigen-specificcytokine-producing cells in recipients after implantation of mice withfree PAE, matrix-embedded PAE, or PAE injection adjacent to emptyGelfoam. There were no significant differences in frequency ofxenoreactive INF-γ and IL-2 producing T-cells between the three groupson day 28. However, rechallenge with matrix-embedded PAE evokes asignificant increase in xenoantigen-specific INF-γ and IL-2 producingT-cells.

FIG. 3B depicts representative ELISPOT wells for one mouse of eachtreatment group 28 days after first implantation and second implantationrespectively. IL-4 production in response to PAE was measured. Thenumber of IL-4 spots in each well was determined by computer-assistedimage analysis.

FIG. 3C graphically depicts that recipients of free PAE exhibited asignificant increased frequency of xenoreactive IL-4 and IL-10 producingT-cells compared to recipients of matrix-embedded PAE on day 28.Rechallenge with free PAE or PAE injection adjacent to empty Gelfoammatrices significantly increased frequency of xenoantigen-specific IL-4and IL-10 producing T-cells on day 128.

Modulation of Effector Cells.

Splenocytes recovered from the recipients were resuspended in FACSbuffer at a concentration of 2×10⁶/ml. Cells were stained with anti-CD4FITC (clone L3T4), antiCD8 FITC (clone Ly-2), anti-CD44 R-PE (cloneLy-24), and anti-CD62L allophycocyanin (clone Ly-22), and isotypecontrols (all BD PharMingen). CD4⁺ and CD8⁺ effector cells expressingCD44^(high) and CD62L^(lOw) were enumerated, as previously described.

Embedding endothelial cells in a three-dimensional biocompatible matrixprevented xenorejection in mice in vivo. To determine the effect ofmatrix embedding on the generation and function of xenoreactive CD4⁺ andCD8⁺ T cells, we measured the number of CD62L^(low)CD44^(high) found inthe spleens of mice treated after implantation of matrix-embeddedporcine aortic endothelial cells, implantation of saline-suspended cellpellets, or as pellets adjacent to empty Gelfoam 28 days followingimplantation (FIG. 4). CD4⁺ and 8⁺ effector cells have been reliablyidentified as CD62L^(low)CD44^(high) cells. The percentage ofCD62L^(low)CD44^(high) cells increased significantly in free porcineaortic endothelial cell-recipients and mice receiving porcine aorticendothelial cells adjacent to empty Gelfoam compared with mice receivingmatrix-embedded porcine aortic endothelial cells; the frequency ofCD4⁺CD62L^(low)CD44^(high) T cells outnumbered CD8⁺ effector cells inall groups (ratio 1.7-2.3).

FIG. 4A graphically plots significantly increased CD4⁺ and CD8⁺ effectorcells in mice receiving free PAE. CD4⁺ effector cells outnumbered CD8⁺T-cells on days 28 and 128. CD4⁺ splenocytes recovered from mice wereanalyzed by flow cytometry using CD62L and CD44 as markers for effectorT cells. Representative plots from mice receiving free (a),matrix-embedded (b), or PAE adjacent to Gelfoam (c) 28 days afterimplantation.

FIG. 4B graphically depicts expansion of effector cells increases afterrechallenge in mice receiving free PAE but not matrix-embedded PAE.Modulation of xenorejection and immunological memory.

Embedding endothelial cells in a three-dimensional biocompatible matrixproduced immunological memory after implantation of non-vascularizedxenogeneic tissue. Th1 cytokines play critical roles in the preventionof xenorejection by down-regulating the Th2-driven humoral responses. Inthis regard, the data demonstrate that tissue engineered endothelialcells can evoke a significant increase of porcine aortic endothelialcell-specific IgG_(2a) antibodies and a significant increase inxenoreactive Th1 producing splenocytes after rechallenge.

One hundred days after the first implantation, the remaining mice ineach group were rechallenged with porcine aortic endothelial cellsidentical to their first encounter. Mice receiving saline-suspended cellpellets or pellets adjacent to empty Gelfoam showed a significantIgG₁-driven porcine aortic endothelial cell-specific antibody responseexceeding the response observed after the first course of implantation(FIG. 5A). Only a weak IgM-antibody release was seen (FIG. 5B). Inmarked contrast, mice receiving matrix-embedded porcine aorticendothelial cells did not show an increase in porcine aortic endothelialcell-specific anti-IgG₁ and IgM levels but exhibited a significantrelease of porcine aortic endothelial cell-specific IgG_(2a) antibodiesthat was absent in the other two mice groups (FIG. 5C).

FIGS. 5A, 5B and 5C graphically depict rechallenge mice (n=12 per groupto day 128, n=6 per group day 156-190 post-implantation) with free PAEor PAE adjacent to Gelfoam significantly increased formation ofPAE-specific IgG₁-antibodies compared to rechallenge withmatrix-embedded PAE (FIG. 5A). Rechallenge has no influence onPAE-specific IgM-formation (FIG. 5B) and there were no significantdifferences of PAE-specific IgG_(2a)-antibodies between the three groups(FIG. 5C).

In line with these results, isolated splenocytes from mice receivingfree porcine aortic endothelial cells or porcine aortic endothelialcell-injections adjacent to empty Gelfoam showed significantly increasedcapability to lyse porcine aortic endothelial cells 28 days afterrechallenge, whereas lysing-capability of splenocytes from micereceiving a second implant of matrix-embedded porcine aortic endothelialcells was significantly weaker than after the first implantation (FIG.6). The frequency of xenoreactive IL-4 and IL-10 producing T cellsincreased significantly in mice after reimplantation of free porcineaortic endothelial cells, the frequency of Th2 producing splenocytesafter rechallenge with matrix-embedded porcine aortic endothelial cellswas unchanged. However, rechallenge with matrix-embedded porcine aorticendothelial cells induced a higher frequency of xenoreactive INF-γ andIL-2 producing splenocytes than after the first course of implantation.

FIG. 6 graphically depicts matrix embedding or MHC II blockade restoreproliferation of mice splenocytes exposed to PAE to unstimulated levels.Mice splenocytes proliferate in response to INF-γ stimulated PAE.Matrix-embedding endothelial cells or presence of MHC II antibodyblocked splenocyte proliferation in response to INF-γ treated PAE by˜79%. Each value represents mean±SD.

Furthermore, 28 days after rechallenge the percentage of CD4⁺ effectorcells further increased in mice receiving free porcine aorticendothelial cells and increased significantly in mice receiving porcineaortic endothelial cells adjacent to empty Gelfoam implants but remainedunchanged in mice receiving matrix embedded porcine aortic endothelialcells. The same pattern was obvious for CD8⁺ effector T cells.

In vitro stimulation of naïve mice splenocytes with PAE revealed asignificantly muted proliferative response of splenocytes when incubatedwith INF-γ stimulated matrix-embedded endothelial cells compared to freeendothelial cells. The presence of MHC II antibody blocked splenocyteproliferation in response to INF-γ-treated PAE by 79% to a levelcomparable to matrix embedded PAE.

Overall, the spleen size in mice receiving matrix-embedded porcineaortic endothelial cells appeared smaller than in the other groups atthe end of the study period (62.9±9.6, 112.7±16.9, 102.5±18.8 mm³;p<0.05).

Thus, cognate interactions between naïve T cells and resting endothelialcells can lead to tolerance in vitro and in vivo. These data documentformation of immunological memory after implantation of non-vascularizedxenogeneic tissue. Immunological memory was characterized by asignificant increase in antigen-specific IgG₁ and IgM levels, lyticactivity of splenocytes and tendency towards increased differentiationinto effector T cells. In contrast, rechallenging mice with matrixembedding of endothelial cells led to a reduced lytic ability ofsplenocytes, frequency of effector CD4⁺ and CD8⁺ T cells was unchanged.Whereas rechallenge with matrix-embedded porcine aortic endothelialcells had no influence on generation of anti-PAE IgG₁ and IgM, IgG_(2a)levels increased significantly.

Modulation of Fractalkine Expression.

Chemokines and adhesion molecules are critical in recruiting circulatingimmune cells into the vessel wall. Fractalkine has both chemoattractiveand adhesive functions and is involved in the pathogenesis ofatherosclerosis, cardiac allograft rejection, glomerulonephritis, andrheumatoid arthritis. We compared expression and secretion offractalkine between free and matrix-embedded human aortic endothelialcells (HAE) via RT-PCR, Western blot, flow-cytometry and ELISA. Adhesionassays were conducted with cytokine-stimulated HAE and ⁵¹Cr labelednatural killer (NK) cells.

HAE were stimulated with 100 U TNFα/ml (Sigma) and 100 U IFN-γ/ml(Roche) at 37° C. in a humidified air atmosphere containing 5% CO₂,conditions demonstrated to result in maximal fractalkine levels incultured endothelial cells.

Flow Cytometry: Endothelial cell monolayers or endothelial cellsmatrix-embedded in Gelfoam were harvested after stimulation with TNFαand IFN-γ for indicated time periods. Media were aspirated and cellswere washed with PBS. Monolayers were incubated in 1 mM PBS/EDTA for 5min, and disrupted by gentle shaking. Gelfoam-grown cells were digestedwith collagenase type I (Worthington Biochemical, NJ), which was shownto have no effect on CX3CL1-expression. Cell-suspensions were washed and3×10⁵ cells fixed in 4% paraformaldehyde for 10 min. After two washingsteps, cells were resuspended in saponin-buffer (0.1% saponin, 0.05%NaN₃ in Hanks' Balanced Salt Solution), centrifuged and the supernatantdecanted. HAE were then incubated with FITC-conjugated mouse anti-humanCX3CL1 (IgG₁, clone 51637, R&D Systems, Minneapolis, Minn.) or a matchedisotype control (clone MOPC-31C, Pharmingen) for 45 min at 4° C. Cellswere then washed and 10⁴ cells were analyzed by flow cytometry using aFACScalibur instrument and CellQuest software.

Western blot analysis: Cell monolayers or cells digested from Gelfoammatrices by collagenase-treatment were washed in PBS buffer and celllysates were prepared by incubation with lysis buffer (20 mM Tris, 150mM NaCl, pH 7.5, 1% Triton X-100, 1% deoxycholate, 0.1% SDS and proteaseinhibitor; Roche). Samples were separated on 4-20% Ready Tris-HCl gels(BioRad Laboratories, Hercules, Calif.). A positive control forfractalkine detection was used, consisting of an 85- to 90-kDa form ofrecombinant human fractalkine lacking the carboxy-terminal 57 aminoacids (R&D Systems). Proteins were then transferred onto PVDF membranes(Millipore, Billerica, Mass.) by using glycin-Tris transfer buffer. Blotmembranes were blocked in Starting Block blocking buffer (Pierce,Rockford, Ill.) for 1 hour. For fractalkine-detection, blocked membraneswere incubated with goat anti-human fractalkine polyclonal antibody (R&DSystems) at a dilution of 1:200 in blocking buffer overnight at 4° C.Membranes were then washed three times at room temperature with washbuffer consisting of PBS with 0.05% Tween 20 and then incubated withsecondary antibody, a rabbit anti-goat IgG conjugated to horseradishperoxidase (Santa Cruz Biotechnology, Santa Cruz, Calif.) at a 1:3.000dilution in blocking buffer for 2 hours at room temperature followed bywashing in five changes of wash buffer. For detection of fractalkinebands, the blot was incubated with chemiluminescence substrate (WesternLightning Chemiluminescence Reagent Plus kit, Perkin-Elmer, Boston,Mass.) according to the manufacturer's instructions followed by exposureto X-ray film (Kodak X-Omat Blue XB-1).

ELISA: Conditioned medium from endothelial cell monolayers orendothelial cells embedded in Gelfoam after cytokine stimulation washarvested for indicated time periods. Secreted fractalkine was detectedwith a commercially available enzyme-linked immunosorbent assay (ELISA)detection kit (R&D Systems). Briefly 96-well Immulon plates (FisherScientific, Pittsburgh, Pa.) were coated overnight at room temperaturewith 100 μl of 4 μg/ml of mouse anti-human fractalkine capture antibodyin PBS. After three washes with wash buffer (PBS-0.05% Tween-20) plateswere blocked for 3 h in 1% bovine serum albumin-5% sucrose in PBS. 100μl of standards (420 ng/ml of recombinant human fractalkine (providedwith kit) was used diluted as twofold serial dilutions in diluentbuffer) or conditioned medium were added, followed by incubationovernight at room temperature. After three washing steps the plate wasincubated with 100 μl of 500 ng/ml mouse anti-human fractalkinedetection antibody in PBS for 2 hours at room temperature followed byincubation with 100 μl of streptavidin conjugated tohorseradish-peroxidase for 30 min at room temperature. Color was thendeveloped by adding 100 μl hydrogen peroxide solution mixed withtetramethylbenzidine (R&D Systems). The optical density was then read ata wavelength of 450 nm.

NK cell-endothelial cell binding assays: HAE were grown to confluence in6-well plates (6×10⁵ cell/well) or embedded in Gelfoam matrices andactivated with 100 U TNFα/ml and 100 U IFN-γ/ml for 20 hours at 37° C.in a humidified air atmosphere containing 5% CO₂ and washed once withPBS. Gelfoam matrices were digested with collagenase type I, cellscounted and plated at a concentration of 6×10⁵ cells/well in 6-wellplates for 1 hour to allow adherence. Isolated NK cells were incubatedwith 10 μCi of ⁵¹Cr/10⁶ NK cells, washed in PBS and then resuspended (510⁵/well) in 400 μl of medium alone or medium containing anti-CX3CR1antibody at 20 μg/ml for 20 min. The NK cell suspension was added to theendothelial monolayer under gentle rocking conditions (10 cycles/min).After 30 min the medium was decanted and the wells were gently washed.Adherent cells were lysed by treating with 1% Triton in PBS. Totalbinding was determined by measuring individual well-associated countsper minutes using a gamma counter. The analyses illustrated wererepresentative of at least three independent experiments.

Matrix-embedding repressed induction of fractalkine mRNA. Whereasresting endothelial cells grown on tissue culture polystyrene plates orwithin a three-dimensional matrix did not express fractalkine,stimulation of HAE grown on tissue culture polystyrene plates with TNFαand IFN-γ induced fractalkine mRNA expression in a time dependentmanner. Fractalkine mRNA in HAE grown on tissue culture polystyreneplates expression peaked at 12 hours stimulation with cells stillexpressing significant amounts of mRNA after stimulation for 24 hours.In contrast, induction of fractalkine mRNA expression was significantlyreduced in matrix-embedded endothelial cells at all time points studied.The maximum was also reached after 12 hours cytokine stimulation but wasonly ˜10% of expression levels in endothelial cells grown to confluenceon tissue culture polystyrene plates (p<0.0001).

Matrix-embedding inhibited fractalkine protein expression in HAE.Western blot analysis revealed lower protein expression levels offractalkine in HAE embedded within Gelfoam matrices compared toendothelial cells grown on tissue culture polystyrene plates. There wasno fractalkine-specific protein band detectable in unstimulatedendothelial cells and in endothelial cells stimulated for 4 hours.Endothelial cells grown on tissue culture polystyrene plates expressedfractalkine after 8 hours of stimulation and exhibited maximalexpression from 16 to 24 hours of stimulation with TNFα and IFN-γ.Protein-expression in matrix-embedded HAE was detectable later (12hours), weaker and disappeared within 24 hours of cytokine stimulation.

In analogy to Western blot results, flow cytometry analysis revealedsignificant higher fractalkine protein expression level in HAE grown ontissue culture polystyrene plates. Whereas maximal expression onmatrix-embedded endothelial cells was reached after 16 hours of cytokinestimulation (22.8±5.7%), endothelial cells grown on tissue culturepolystyrene plates reached a maximal and significant increasedfractalkine expression after 20 hours stimulation with TNFα and IFN-γ(76.5±8.6%; p<0.0001).

Experimental data indicate a reduced secretion of fractalkine fromcytokine stimulated matrix-embedded endothelial cells. Fractalkinelevels were also measured as cumulative levels of soluble fractalkinereleased into the endothelial culture supernatants by ELISA. Levels ofsoluble fractalkine paralleled those in Western blot and flow cytometryanalysis: fractalkine secreted from HAE grown on tissue culturepolystyrene plates significantly exceeded levels secreted bymatrix-embedded HAE (32.2±2.4 vs. 13.8±1.7 pg/ml after 24 hours ofculture; p<0.0002).

Experimental data indicate a reduced adhesion of NK cells tomatrix-embedded endothelial cells. To study the functional relevance ofour finding, an adhesion assay with ⁵¹Cr labeled NK cell andcytokine-stimulated HAE grown on tissue culture polystyrene plates ormatrix-embedded was performed next. As revealed by gamma-counting,significantly more NK-cells adhered to allogeneic HAE grown on tissueculture polystyrene plates than embedded within Gelfoam (6335±420 vs.1735±135 cpm; p<0.0002; FIG. 5). The importance of fractalkineexpression for NK cell adhesion to activated endothelial cells could bedemonstrated as addition of 20 μg/ml anti-CX3CL1 significantly augmentedadhesion of NK cells to cytokine stimulated HAE by ˜74% (p<0.005 vs.without anti-CX3CL1). NK cells did not adhere to tissue culturepolystyrene plates or Gelfoam alone.

Modulation of the Immune Response in Heightened Immune Reactivity Mice.

Endothelial cell injections induced antibody formation in mice. In naïveB6 mice three serial subcutaneous injections of 5×10⁵ PAE raisedcirculating endothelial cell-specific IgG₁ (2210±341 vs. 53±12 meanfluorescence intensity (MFI); p<0.0001) and IgM antibodies compared tosaline injections (136±39 vs. 49±14 MFI; p<0.02). There were noPAE-specific IgG_(2a) antibodies detectable in serum of either mousegroups (data not shown) 42 days after first injection of PAE.

Matrix-embedded endothelial cells prevented humoral immune reactivity.Implantation of matrix-embedded xenogeneic endothelial cells, in markedcontrast to implantation of free cells, failed to induce a significanthumoral immune response in naïve mice (d 42, IgG₁: 210±102 vs. 735±327MFI; p<0.001; IgM: 60±11 vs. 299±51 MFI; p<0.001; FIGS. 7A and 7B).Injection of free PAE in pre-sensitized serially challenged miceresulted in an elevated humoral immune response with a pronouncedincrease in IgG₁ antibody-levels (3795±448 MFI; p<0.0002 vs. naïve mice)and slight increase in PAE-specific IgM (164±28 MFI). In markedcontrast, implantation of matrix-embedded PAE in pre-sensitized seriallychallenged mice did not increase PAE-specific antibodies: moreoverantibody-levels specific for the injected PAE slowly decreased with time(IgG₁: 1578±334 MFI; p<0.0005 vs. free. PAE; IgM: 69±5 MFI; p<0.01 vs.free PAE; FIGS. 7A and 7B). There was no increase in PAE-specificIgG_(2a)-antibodies in the four treatment groups (data not shown)supporting previous reports of a dominating Th2 response inxenografting.

FIGS. 7A and 7B graphically depict circulating PAE-specific IgG₁ (FIG.7A) and IgM (FIG. 7B) in naïve and pre-sensitized serially challengedmice after subcutaneous implantation of non-embedded or matrix-embeddedPAE. Graphic depiction of results from all mice (n=112/group to day 70,n=6/group day 71-132 post-implantation) demonstrates significantdifferences between matrix-embedded and free PAE implantation.Antibody-levels after implantation of matrix-embedded PAE slowlydiminish. Data are expressed as mean values±SD.

Matrix-embedded endothelial cells are poor inducers of cellularimmunity. ELISPOT-analysis revealed a high frequency of xenogeneicT-helper cell (Th)2-cytokine (IL-4, IL-10) producing splenocytes innaïve and pre-sensitized serially challenged mice 90 days afterimplantation of free but not after implantation of matrix-embedded PAE.The frequency of xenoreactive splenocytes in pre-sensitized seriallychallenged mice exceeded xenoreactive splenocyte activation anddifferentiation in naïve mice receiving free PAE (IL-4: 907±59 vs.680±129; p<0.02; IL-10: 1096±94 vs. 888±151 number of spots; p<0.02;FIGS. 8A and 8B). Yet, compared to implantation of matrix-embedded PAEin naive mice, implantation of matrix-embedded PAE in pre-sensitizedserially challenged mice elicited only a slight increase in IL-4 (322±75vs. 199±99 number of spots; p<0.05; p<0.0005 vs. free PAE; FIG. 8A) butnot in IL-10 producing xenoreactive splenocytes (403±142 vs. 451±135number of spots; p=0.27; p<0.001 vs. free PAE; FIG. 8B). Significantlyfewer Th2-cytokine producing splenocytes were present in pre-sensitizedserially challenged mice receiving matrix-embedded PAE compared to naïvemice receiving free PAE (p<0.001). The frequency of Th1-cytokine (IFN-γand IL-2) producing splenocytes did not differ significantly between thefour treatment groups again supporting a predominant Th2-role inxenoreactivity (data not shown).

FIGS. 8A and 8B graphically depict the frequencies of xenoreactivecytokine-producing cells in recipients after implantation of free PAE ormatrix-embedded PAE in naïve and pre-sensitized serially challengedmice. Data are expressed as mean values±SD. Naïve and pre-sensitizedserially challenged recipients of free PAE exhibited significantincreased frequencies of IL-4 (FIG. 8A) and IL-10 (FIG. 8B) producingsplenocytes compared to recipients of matrix-embedded PAE.

The increase in cytokine-producing splenocytes in mice receivingnon-embedded PAE was paralleled by an increase of CD4⁺ and CD8⁺ effectorT cells over time (CD4⁺: 44±2 naïve mice, 54±4% pre-sensitized mice,p<0.05; CD8⁺: 20±2; 21±2%; FIGS. 9A and 9B). Accordingly,differentiation of T cells into CD44^(high)/CD62L^(low) T cells wassignificantly muted in naive and pre-sensitized serially challenged miceexposed to matrix-embedded PAE (CD4⁺: 22±2 naïve mice, 21±3%pre-sensitized mice; p<0.01 vs. free PAE; CD8⁺: 12±2; 14±3%; p<0.02 vs.free PAE; FIGS. 9A and 9B). CD4⁺ outnumbered CD8⁺ effector T cells1.7-2.6 in all treatment groups. A strong correlation was noted betweenthe frequency of Th2-cytokine producing splenocytes and extent of T celldifferentiation cells into CD4⁺CD44^(high)/CD62L^(low) (IL-4: r=0.81;p<0.0001; IL-10 r=0.88; p<0.0001; FIG. 10) andCD8⁺CD44^(high)/CD62L^(low) effector cells (IL-4: r=0.79; p<0.0001;IL-10 r=0.86; p<0.0001) across all treatment groups on day 132.

FIGS. 9A and 9B graphically depict significantly increased CD4⁺ (FIG.9A) and CD8⁺ (FIG. 9B) effector cells in mice receiving free PAE.Splenocytes recovered from mice were analyzed by flow-cytometry usingCD62L and CD44 as markers for effector T cells. No difference betweennaïve and pre-sensitized serially challenged mice when endothelial cellsare matrix-embedded. Data are expressed as mean values±SD.

FIGS. 10A and 10B are Spearman correlations of the frequencies ofTh2-cytokine producing splenocytes and the extent of T celldifferentiation into effector cells. FIG. 10A graphically depicts thefrequency of IL-2 cytokines. FIG. 10B graphically depicts the frequencyof IL-10 cytokines. The correlations suggest that cytokine levelscorrelate linearly with effector T cell induction. Area of the densityellipse represents the 95% confidence interval.

Matrix-embedded endothelial cells are shielded from lytic damage. Theability of host lymphocytes to damage xenogeneic endothelial cells wascharacterized on day 70 and day 132. Calcein release plateaued ateffector: target ratios of 25:1. For this ratio, endothelial cell damagewas 1.6 fold higher in naïve mice and 1.7 fold higher in pre-sensitizedmice when receiving non-embedded in place of matrix-embedded PAE on d70(p<0.001). These ratios increased to 1.9 and 2.3 respectively after 132days (p<0.0005; FIG. 11). Of note, the extent of endothelial damage inpre-sensitized mice receiving matrix-embedded PAE was significant lowerwhen compared to naïve mice receiving free PAE (20.9±2.3 vs. 37.1±3.4%AFU; p<0.001; FIG. 11).

The ability of host lymphocytes to damage xenogeneic endothelial cellswas characterized on day 70 and day 132. FIG. 11 graphically depicts thedegree of damage to endothelial cells in naïve and pre-sensitized micewhen the endothelial cells are free or matrix embedded. Endothelialdamage via lysis is significantly reduced in naïve and pre-sensitizedmice receiving matrix-embedded compared to free PAE. 2×10⁴ PAE werelabeled with calcein and incubated with 5×10⁵ splenocytes isolated after70 and 132 days respectively.

Calcein release plateaued at effector: target ratios of 25:1. For thisratio, endothelial cell damage was 1.6 fold higher in naïve mice and 1.7fold higher in pre-sensitized mice when receiving non-embedded in placeof matrix-embedded PAE on day 70 (p<0.001). These ratios increased to1.9 and 2.3 respectively after 132 days (p<0.0005; FIG. 11). Of note,the extent of endothelial damage in pre-sensitized mice receivingmatrix-embedded PAE was significant lower when compared to naïve micereceiving free PAE (20.9±2.3 vs. 37.1±3.4% AFU; p<0.001; FIG. 11).

Modulation of Dendritic Cell Maturation.

Dendritic cells are antigen-presented cells that have the unique abilityto both initiate and regulate immune responses. Mature dendritic cellspromote T cell differentiation into effector and memory cells whereasimmature dendritic cells present (self-)antigens in a tolerogenicfashion. Dendritic cells are implicated in a variety ofendothelial-mediated diseases, and activated endothelial cells inducetheir maturation. Because dendritic cells are critical in immunereactivity, it follows that endothelial cell-driven dendritic cellmaturation is dependent on endothelial cell-matrix contact.

Preparation, culture and maturation of dendritic cells: Peripheral bloodwas collected from healthy volunteers and fractionated over Ficoll-Paque(Sigma Chemicals, St. Louis, Mo.) by a standard procedure. To derivedendritic cells, total peripheral blood monocytic cells (PBMC) werecultured at 2×10⁶ cells/ml in complete media (RPMI 1640, 10%heat-inactivated calf serum, 0.1 mM sodium pyruvate (Life Technologies))for 1.5 hours in tissue culture flasks. Following incubation,nonadherent cells were removed by extensive washing with a 1× solutionof HBSS (Life Technologies). The remaining adherent cells were thencultured in complete media containing 20 ng/ml interleukin (IL)-4 and 20ng/ml GM-CSF (Peprotech, Rocky Hill, N.J.) for 5 days in a CO₂ incubatorat 37° C. The resulting cells were semi- to nonadherent and MHCII^(low)/CD14^(−/low)/CD83⁻ (data not shown).

For further maturation, adherent and nonadherent dendritic cells wereharvested, extensively washed, counted and 5×10⁵ dendritic cells werestimulated with a cytokine cocktail (10 ng/ml IL-1β, 1000 U/ml IL-6, 20ng/ml IL-4, GM-CSF, and TNF-α; all Preprotech), 1.5×10⁵ HAE or 1.5×10⁵PAE for 48 hours. Endothelial cells were either presented as suspensionsafter grown to confluence on tissue culture plates or surface adherentembedded within Gelfoam matrices. Every assay was repeated at least fourtimes. After maturation, dendritic cells were isolated from anycontaminating endothelial cells with magnetic bead-labeled CD1aantibodies (Miltenyi, Bergisch-Gladbach, Germany). Flow cytometryanalysis revealed 98% purity of the isolated DC (data not shown).

Real-time PCR: Total RNA was extracted from isolated dendritic cells andthe remaining endothelial cells using the RNeasy Mini Kit (Qiagen,Valencia, Calif.) according to the manufacturer's instructions.Complementary DNA was synthesized using TaqMan reverse transcriptionreagents from Applied Biosystems (Foster City, Calif.). Real-time PCRanalysis was performed with an Opticon Real Time PCR Machine (MJResearch, Waltham, Mass.) using SYBR Green PCR Master Mix (AppliedBiosystems) and selected primers. Data from the reaction were collectedand analyzed by the complementary Opticon computer software. Relativequantification of gene expression were calculated with standard curvesand normalized to GAPDH.

Flow cytometry: Dendritic cell or endothelial cell suspensions werewashed and 3×10⁵ cells were resuspended in FACS buffer (PBS containing0.1% BSA and 0.1% sodium azide; Sigma Chemicals). Standard flowcytometric analysis assessed surface expression of various markers. Thefollowing monoclonal antibodies directly conjugated with phycoerythrin(PE) or fluorescein isothiocyanate (FITC) were used in single-color flowcytometric analysis: PE-CD1a (clone HI149, IgG₁), FITC-CD3 (clone UCHT1,IgG₁), PE-CD14 (clone TÜK4, IgG_(2a)), PE-CD31 (clone WM59, IgG₁),FITC-CD40 (clone 5C3, IgG₁), FITC-CD54 (clone 15.2, IgG₁), FITC-CD80(clone BB1, IgM), FITC-CD83 (clone HB15e, IgG₁), FITC-CD86 (clone 2331,IgG₁), FITC-CD106 (clone 51-10C9, IgG₁), FITC-HLA-DP,DQ,DR (cloneCR3/43, IgG₁), FITC-Toll-like receptor (TLR)2 (clone TL2.3, IgG_(2a)),and FITC-TLR4 (clone HTA125, IgG_(2a)). Appropriate isotype controlantibodies (mouse PE-IgG₁, PE-IgG2a, FITC-IgG₁, FITC-IgG_(2a), FITC-IgM)were used respectively. Antibodies were purchased from DakoCytomation(Carpinteria, Calif.), Serotec (Raleigh, N.C.) or PharMingen (San Diego,Calif.). After staining, cells were washed and fixed in 1%paraformaldehyde before analysis on a FACScalibur instrument andCellQuest software (Becton Dickinson, Mountain View, Calif.).

Endocytic activity: Endocytic activity of dendritic cells was measuredby uptake of FITC-conjugated dextran (MW 40.000; Molecular Probes,Eugene, Oreg.) as previously described. Briefly, dendritic cells atvarious states of maturation were incubated in complete media with 1mg/ml FITC-conjugated dextran for 1 hour at 37° C. to measure specificuptake, or at 4° C. to measure nonspecific binding. Cells were thenwashed extensively and analyzed by flow cytometry as described above.

Mixed lymphocyte reaction assay: CD3⁺ T-cells from an unrelated donorwere prepared from total PBMC by negative selection using antibodydepletion and magnetic beads according to the manufacturer's instruction(Dynal Biotech, Lake Success, N.Y.). The nonmagnetic fraction containedgreater than 95% CD3⁺ T-cells, as assessed by flow cytometry. 2×10⁵ CD3⁺T-cells/well were seeded in 96-well round-bottom plates. Purifiedcytokine- or endothelial cell-matured dendritic cells were γ-irradiated(3000 rad from a ¹³⁷Cs source) and added to T-cells at 1×10⁴, 4×10³, or2×10³ cells/well to give final ratios of 1:20, 1:50, or 1:100DC:T-cells. On day 5, 1 μCi of ³H-thymidine (Perkin-Elmer, Boston,Mass.) was added to each well. Cells were harvested 18 hours later and³H-thymidine uptake quantified using a Packard TopCount γ-counter (GMI,Ramsey Mich.).

Western Blot: After separation from dendritic cells, endothelial cellswere washed in PBS buffer and cell lysates were prepared by incubationwith lysis buffer (20 mM Tris, 150 mM NaCl, pH 7.5, 1% Triton X-100, 1%deoxycholate, 0.1% SDS and protease inhibitor; Roche, Indianapolis,Ind.). Samples were separated on 4-20% Ready Tris-HCl gels (BioRadLaboratories, Hercules, Calif.). Proteins were then transferred ontoPVDF membranes (Millipore, Billerica, Mass.) using glycin-Tris transferbuffer. Jurkat (TLR2) or HL-60 whole cell lysates (TLR4, both Santa CruzBiotechnologies, Santa Cruz, Calif.) served as controls. Membranes wereblocked in Starting Block blocking buffer (Pierce, Rockford, Ill.) for 1hour. Blocked membranes were incubated with rabbit anti-human TLR2(dilution 1:250 in blocking buffer) or TLR4 antibodies (dilution 1:200,both Santa Cruz Biotechnologies) overnight at 4° C. Membranes were thenwashed three times at room temperature with wash buffer consisting ofPBS with 0.05% Tween 20 and then incubated with secondary antibody, agoat anti-rabbit IgG conjugated to horseradish peroxidase (Santa CruzBiotechnology, Santa Cruz, Calif.) at a 1:1.000 dilution in blockingbuffer for 2 hours at room temperature followed by washing in fivechanges of wash buffer. For detection of TLR bands, the blot wasincubated with chemiluminescence substrate (Western LightningChemiluminescence Reagent Plus kit; Perkin-Elmer) according to themanufacturer's instructions followed by exposure and analysis on a FluorChem SP (Alpha Innotech, San Leandro, Calif.).

Non-adherent endothelial cells directed maturation of monocyte-deriveddendritic cells. In line with previous observations, monocytesdifferentiated into immature dendritic cells after 5 days of culture inGM-CSF and IL-4 (data not shown). Prolonged cytokine-stimulation withIL-1β, TNF-α, and IL-6 for 48 hours upregulated costimulatory (CD40: 2.3fold compared to immature dendritic cells, CD80: 1.9 fold, CD86: 1.6fold) and HLA-DR molecules (1.5 fold) together with expression of CD83(2.2 fold) as an established dendritic cells-maturation marker. Exposureto saline suspensions of allo- and xenogeneic endothelial cells aftergrowth to confluence in tissue culture plates induced full maturation ofmonocyte-derived dendritic cells to a similar degree as prolongedtreatment with a cytokine cocktail. HAE or PAE alone induced dendriticcell costimulatory molecule expression with increases in CD40 (HAE: 2.1fold, PAE: 2.5 fold compared to immature dendritic cells), CD80 (2.1fold, 2.3 fold; p<0.05 vs. cytokine-stimulation), CD86 (1.6 fold, 1.7),HLA-DR (1.7 fold, 2.2 fold; p<0.05 vs. HAE, p<0.002 vs.cytokine-stimulation), and CD83 (2.6 fold; p<0.05 vs. cytokinestimulation, 3.2 fold; p<0.02 vs. HAE, p<0.001 vs.cytokine-stimulation).

In a similar fashion, dendritic cell TLR2 and 4 expression wereupregulated upon exposure to saline suspensions of HAE (1.5 and 2.5 foldrespectively) to a similar or greater extent than cytokine stimulation(1.5 fold for both TLR compared to immature dendritic cells). Thiseffect was even more pronounced after co-incubation of dendritic cellswith non-adherent xenogeneic PAE (TLR2: 2.4 fold; p<0.05 vs. cytokine-and HAE-stimulated, TLR4: 3.0 fold; p<0.05 vs. HAE, p<0.001 vs.cytokine-stimulation). Similar results could be obtained for mRNAtranscript levels. Additionally, dendritic cells matured with cytokinesor non-adherent endothelial cells displayed significant upregulation ofIL12 p40 mRNA (immature: 0.03±0.02 relative units (RU),cytokine-stimulated: 0.23±0.03 RU, p<0.002, HAE-stimulated: 0.31±0.05RU, p<0.001, PAE-stimulated: 0.28±0.03, p<0.002).

Incubation with substrate-adherent endothelial cells resulted inincomplete dendritic cell-maturation and sustains endocytic activity. Inmarked contrast to co-culture with non-adherent endothelial cells,co-culture of dendritic cells with substrate-adherent HAE and PAEembedded within a three-dimensional matrix restricted dendritic cellmaturation: these dendritic cells displayed only weak upregulation ofCD40 (substrate-adherent HAE: 1.5 fold compared to immature DC, p<0.02vs. non-adherent HAE, substrate-adherent PAE: 1.3 fold, p<0.002 vs.non-adherent PAE), CD80 (substrate-adherent HAE and PAE: 1.3 fold,p<0.005 vs. non-adherent EC), CD86 (substrate-adherent HAE: 1.1 fold,PAE: 1.2 fold, both p<0.005 vs. non-adherent EC), CD83(substrate-adherent HAE: 1.5 fold, p<0.001 vs. non-adherent HAE, PAE:1.4 fold, p<0.0002 vs. non-adherent PAE), and TLR4 (substrate-adherentHAE: 1.5 fold, PAE: 1.3 fold, both p<0.005 vs. non-adherent endothelialcells). Co-incubation with substrate-adherent endothelial cells failedto induce HLA-DR and TLR expression on dendritic cells at all (p<0.005).Incubation with empty Gelfoam matrices alone had no effect on maturationof monocyte-derived dendritic cells (data not shown). Real-time PCRanalysis revealed the same pattern of incomplete maturation whendendritic cells were exposed to substrate-adherent allo- and xenogeneicendothelial cells. Induction of IL12 p40 was similarly significantlyweaker when dendritic cells had been matured with substrate-adherentendothelial cells (HAE-stimulated: 0.06±0.01, p<0.005, PAE-stimulated0.07±0.02, p<0.02).

Immature dendritic cells efficiently captured antigen and exhibited ahigh level of endocytosis. FITC-conjugated dextran uptake increased whenmonocytes were cultured for 3 and 5 days in GM-CSF and IL-4 (423.3±121.8mean fluorescence intensity (MFI), 239.8±42.8 MFI, p<0.0001). Maturationis typically accompanied by concomitant increase in antigen presentingfunction and reduced capacity for antigen capture via endocyticactivity. Dextran uptake typically decreases with continuedcytokine-stimulation (89.7±14.7 MFI, p<0.0001 vs. d5) and withco-incubation with non-adherent HAE (92±20.3 MFI) or PAE (82.4±16.5MFI). In marked contrast, dendritic cells retained their endocyticactivity when endothelial cells were presented in a substrate-adherentthree-dimensional state and dextran uptake was markedly increased(substrate-adherent HAE: 203.2±11.3 MFI, p<0.05 vs. d5, p<0.0001 vs.non-adherent HAE; substrate-adherent PAE: 254.3±32 MFI, p<0.0001 vs.non-adherent PAE).

Dendritic cells exhibited reduced T-cell proliferation activity aftercultivation with substrate-adherent endothelial cells. The ability topromote T cell differentiation into effector and memory cells is animportant functional marker for the maturation grade of dendritic cells.Whereas cytokine-treated and non-adherent endothelial cell exposeddendritic cells induced T-cell proliferation over the full spectrum ofdendritic cell:T-cell ratios tested (74789±1777, HAE: 97522±1630, andPAE: 101616±4302 cpm) this ability was significantly muted in dendriticcells co-incubated with substrate-adherent HAE (18320±1000 cpm, p<0.002)and PAE (20080±683 cpm, p<0.0001).

Activation of substrate-adherent endothelial cells was reduced whenco-cultured with dendritic cells. Real-time PC, flow-cytometry andWestern blot analysis revealed reduced activation of HAE and PAE afterco-culture for 2 days with dendritic cells. After magnetic-bead basedisolation of dendritic cells, the remaining cells were greater than 95%pure for the endothelial-cell specific marker CD31 (data not shown).Real-time PCR analysis demonstrated reduced mRNA expression levels foradhesion molecules, CD58, HLA-DR and TLR-molecules on substrate-adherentHAE when compared to their non-adherent counterparts. ReducedmRNA-expression levels translated into reduced surface and intracellularexpression with 3.6 fold lower expression of ICAM-1 onsubstrate-adherent when compared to non-adherent HAE (1.3 fold decreasefor PAE), 4.9 fold decrease of VCAM-1 for HAE (PAE: 2.7 fold), and 16fold decrease of HLA-DR for HAE (PAE: 23 fold decrease). Densitometryanalysis of Western blots revealed increased TLR2 (HAE: 1.5 foldincrease, PAE: 1.6 fold increase; p<0.05) and TLR4 expression (HAE: 2.3fold increase, PAE: 2 fold increase; p<0.01) in non-adherent endothelialcells when compared to substrate-adherent endothelial cells afterco-incubation with dendritic cells for 48 hours.

Thus, whereas non-adherent endothelial cells induced maturation ofmonocyte-derived dendritic cells to an extent similar to that seen witha cytokine-cocktail, co-incubation with substrate-adherent endothelialcells induced only minor upregulation of mRNA transcript and proteinlevels of adhesion, costimulatory and HLA-DR molecules on dendriticcells. Dendritic cells co-incubated with substrate-adherent endothelialcells also lacked upregulation of IL12 mRNA and CD83 expression thatserve as direct maturation markers. The immature state of dendriticcells after co-cultivation with substrate-adherent endothelial cells wasmirrored by sustained ability to uptake dextran. Functionally, whereasdendritic cells exposed to non-adherent endothelial cells displayedenhanced T-cell stimulatory activity in mixed lymphocyte reactions,T-cell proliferation after exposure to substrate-adherent endothelialcell-matured dendritic cells was significantly weaker.

Further Experiments Effects on Immune Response

Treatment of transplantation rejection: A population of normal (notimmune compromised) organ transplant recipients will be identified. Thepopulation will be divided into three groups, one of which will receivean effective amount of the implantable material of the present inventionprior to receipt of a transplant organ. A second group will receive aneffective amount of implantable material of the present inventioncoincident with receipt of a transplant organ. A third group will notreceive the implantable material of the present invention, but willreceive a transplant organ. Reduction of and/or amelioration of animmune response and/or an inflammatory response will be monitored overtime by evaluating the proliferation of T-cell lymphocytes and B-celllymphocytes in serum samples and by monitoring the duration oftransplant organ acceptance. It is expected that candidates receiving aneffective amount of the implantable material of the present inventionwill display a reduction in proliferation of lymphocytes and/or anincrease in the duration of transplant organ acceptance.

Treatment of autoimmune disease: A population of patients diagnosed withan autoimmune disease will be identified. The population will be dividedinto two groups, one of which will receive an effective amount of theimplantable material of the present invention. Reduction of and/oramelioration of an autoimmune response and/or an inflammatory responsewill be monitored over time by evaluating the proliferation of T-celllymphocytes and B-cell lymphocytes in serum samples and by monitoringthe intensity and duration of symptoms associated with the autoimmunedisease. It is expected that candidates receiving an effective amount ofthe implantable material of the present invention will display areduction in proliferation of lymphocytes and/or a reduction in thefrequency and/or intensity of symptoms.

1. A method of reducing an immune response or an inflammatory reaction,comprising the step of: providing to a recipient an implantable materialcomprising a biocompatible matrix; and, anchored or embedded endothelialcells, endothelial-like cells, or analogs thereof, wherein saidimplantable material is provided to said recipient in an amountsufficient to reduce the immune response or inflammatory reaction insaid recipient.
 2. The method of claim 1 wherein the providing step isprior to, coincident with, or subsequent to administration to saidrecipient of one or more doses of a cell, tissue or organ from asyngeneic or non-syngeneic donor.
 3. The method of claim 1 wherein theproviding step is prior to, coincident with, or subsequent to occurrenceof the immune response or inflammatory reaction.
 4. A method of inducingacceptance in a patient, comprising the step of: providing animplantable material comprising a biocompatible matrix and anchored orembedded endothelial cells, endothelial-like cells, or analogs thereof,prior to, coincident with, or subsequent to transplantation ofautograft, xenograft or allograft cells, tissue or organ in said patientin an amount effective to induce acceptance in said patient.
 5. A methodof reducing donor antigen immunogenicity, comprising the step of:providing an implantable material comprising a biocompatible matrix andanchored or embedded endothelial cells, endothelial-like cells, oranalogs thereof prior to, coincident with, or subsequent to introductionof said donor antigen to a recipient in an amount effective to reducedonor antigen immunogenicity.
 6. The method of claim 1 wherein saidproviding step occurs prior to, coincident with, or subsequent toadministration to said recipient of an immunosuppressive agent.
 7. Themethod of claim 6 wherein said immunosuppressive agent resides in saidimplantable material during coincident administration.
 8. The method ofclaim 5 wherein said donor and recipient are the same.
 9. The method ofclaim 1 wherein said recipient has an autoimmune disease.
 10. Animplantable material suitable for use with claim
 1. 11. The implantablematerial of claim 10 wherein the endothelial-like cells or analogs arenon-endothelial cells.
 12. The implantable material of claim 10 whereinthe cells are autogenic, allogenic, xenogenic or genetically-modifiedvariants of any one of the foregoing.
 13. The implantable material ofclaim 10 wherein the cells are vascular endothelial cells.
 14. A methodof transplanting to a recipient a syngeneic or non-syngeneic cell,tissue or organ transplant, comprising the step of: providing to saidrecipient an implantable material comprising a biocompatible matrix andanchored or embedded endothelial cells, endothelial-like cells, oranalogs thereof, prior to, coincident with, or subsequent totransplantation such that said transplanted syngeneic or non-syngeneiccell, tissue or organ is not rejected by said recipient.
 15. The methodof claim 14 wherein said transplanted cell, tissue or organ comprisesnon-endothelial cells.
 16. The method of claim 5 wherein said donorantigen comprises a non-endothelial cell antigen.
 17. The method ofclaim 14 further comprising the step of administering animmunosuppressive agent prior to, coincident with, or subsequent totransplantation.
 18. A cell suitable for use with the implantablematerial of claim
 1. 19. The cell of claim 18 wherein saidendothelial-like cell or its analog is a non-endothelial cell.
 20. Thecell of claim 18 wherein said analog is non-natural.
 21. The cell ofclaim 18 wherein said cell, when anchored to or embedded within abiocompatible matrix, reduces a recipient's humoral or cellular immuneresponse to a non-syngeneic donor cell, tissue or organ.
 22. The cell ofclaim 18 wherein said cell, when anchored to or embedded within abiocompatible matrix, exhibits diminished immunogenicity.
 23. The cellof claim 22 wherein said diminished immunogenicity is reduced expressionof MHC or reduced capacity to bind, activate or promote maturation ofinnate immune cells, when anchored to or embedded within a biocompatiblematrix, wherein said innate immune cells are selected from the groupconsisting of NK cells, dendritic, cells, monocytes, and macrophages.24. The cell of claim 18 wherein said cell, when anchored to or embeddedwithin a biocompatible matrix, exhibits reduced expression of MHC,costimulatory molecules or adhesion molecules.
 25. The cell of claim 18wherein said cell, when anchored to or embedded within a biocompatiblematrix and co-cultured with a dendritic cell, inhibits expression bysaid dendritic cell of HLA-DR, IL12, Toll-like receptor or CD83;promotes uptake of dextran by said dendritic cell; or blocks dendriticcell-induced lymphocyte proliferation; or when co-cultured with adaptiveimmune cells inhibits proliferation, activation or differentiation ofsaid cells, wherein adaptive immune cells are selected from the groupconsisting of B-lymphocytes and T-lymphocytes.
 26. An implantablematerial comprising a biocompatible matrix and the anchored or embeddedendothelial cell, endothelial-like cell, or analog thereof of claim 22.27. An implantable material comprising a biocompatible matrix and theanchored or embedded endothelial cell, endothelial-like cell, or analogthereof of claim
 24. 28. An implantable material comprising abiocompatible matrix and the anchored or embedded endothelial cell,endothelial-like cell, or analog thereof of claim
 25. 29. A cell bankcomprising the cell of claim
 18. 30. A bank of implantable materialcomprising the implantable material of claim
 10. 31. A bank ofimplantable material, wherein said implantable material comprises abiocompatible matrix and the anchored or embedded endothelial cell,endothelial-like cell, or analog thereof of claim
 22. 32. A bank ofimplantable material, wherein said implantable material comprises abiocompatible matrix and the anchored or embedded endothelial cell,endothelial-like cell, or analog thereof of claim
 24. 33. A bank ofimplantable material, wherein said implantable material comprises abiocompatible matrix and the anchored or embedded endothelial cell,endothelial-like cell, or analog thereof of claim
 25. 34. Theimplantable material of claim 10 wherein said implantable material is asolid or non-solid.
 35. The implantable material of claim 10 whereinsaid implantable material is provided to the recipient by implantation,injection or infusion.
 36. An implantable material for reducing animmune response to a non-syngeneic cell, tissue or organ, wherein saidimplantable material comprises: a biocompatible matrix; and, anchoredthereto or embedded therein, endothelial cells, endothelial-like cells,or analogs thereof; or tissue, or organ, or a segment thereof; whereinan effective amount of said implantable material reduces the recipient'simmune response to said non-syngeneic cell, tissue or organ.
 37. Theimplantable material of claim 36 wherein said non-syngeneic cell, tissueor organ is that of the recipient suffering from an autoimmune disease.38. A method of reducing an immune response or an inflammatory reactionresulting from exposure to an exogenous immunogen, comprising the stepof: providing to a recipient an implantable material comprising abiocompatible matrix; and, anchored or embedded endothelial cells,endothelial-like cells, or analogs thereof, wherein said implantablematerial is provided to said recipient in an amount sufficient to reducethe immune response or inflammatory reaction in said recipient resultingfrom exposure to said exogenous immunogen.
 39. The method of claim 38wherein the providing step is prior to, coincident with, or subsequentto occurrence of the immune response or inflammatory reaction.
 40. Themethod of claim 38 wherein said exogenous immunogen is naturallyoccurring.
 41. The method of claim 38 wherein said exogenous immunogenis selected from the group consisting of pharmaceutical agents, toxins,surgical implants, infectious agents and chemicals.
 42. The method ofclaim 38 wherein said exogenous immunogen is an exogenous stimulusselected from the group consisting of environmental stress, injury andexposure.